Oligonucleotide comprising an inosine for treating DMD

ABSTRACT

The invention provides an oligonucleotide comprising an inosine, and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-m RNA exon or at least part of a non-exon region of a dystrophin pre-m RNA said part being a contiguous stretch comprising at least 8 nucleotides. The invention further provides the use of said oligonucleotide for preventing or treating DMD or BMD.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/694,980, filed Nov. 25, 2019, which is a continuation of U.S.application Ser. No. 15/468,239, filed Mar. 24, 2017, now U.S. Pat. No.10,533,171, which is a continuation of U.S. application Ser. No.15/168,662, filed May 31, 2016, which is a continuation of U.S.application Ser. No. 14/678,517, filed Apr. 3, 2015, which is acontinuation of U.S. application Ser. No. 13/266,110, filed Oct. 24,2011, which is a 371 of PCT International Application No.PCT/NL2010/050230, filed Apr. 26, 2010, which claims priority toEuropean application No. 09158731.1, filed Apr. 24, 2009 and U.S.provisional application No. 61/172,506, filed Apr. 24, 2009, the entirecontents of each of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The attached Sequence Listing, titled “0105_09_US1CN5_SL.txt”, createdon May 4, 2021 and 140 KB in size, is herein incorporated by referencein its entirety.

FIELD OF THE INVENTION

The invention relates to the fields of molecular biology and medicine.

BACKGROUND OF THE INVENTION

A muscle disorder is a disease that usually has a significant impact onthe life of an individual. A muscle disorder can have either a geneticcause or a non-genetic cause. An important group of muscle diseases witha genetic cause are Becker Muscular Dystrophy (BMD) and DuchenneMuscular Dystrophy (DMD). These disorders are caused by defects in agene for a muscle protein.

Becker Muscular Dystrophy and Duchenne Muscular Dystrophy are geneticmuscular dystrophies with a relatively high incidence. In both Duchenneand Becker muscular dystrophy the muscle protein dystrophin is affected.In Duchenne dystrophin is absent, whereas in Becker some dystrophin ispresent but its production is most often not sufficient and/or thedystrophin present is abnormally formed. Both diseases are associatedwith recessive X-linked inheritance. DMD results from a frameshiftmutation in the DMD gene. The frameshift in the DMD gene's transcript(mRNA) results in the production of a truncated non-functionaldystrophin protein, resulting in progressive muscle wasting andweakness. BMD occurs as a mutation does not cause a frame-shift in theDMD transcript (mRNA). As in BMD some partly to largely functionaldystrophin is present in contrast to DMD where dystrophin is absent, BMDhas generally less severe symptoms then DMD. The onset of DMD is earlierthan BMD. DMD usually manifests itself in early childhood, BMD in theteens or in early adulthood. The progression of BMD is slower and lesspredictable than DMD. Patients with BMD can survive into mid to lateadulthood. Patients with DMD rarely survive beyond their thirties.

Dystrophin plays an important structural role in the muscle fiber,connecting the extracellular matrix and the cytoskeleton. The N-terminalregion binds actin, whereas the C-terminal end is part of the dystrophinglycoprotein complex (DGC), which spans the sarcolemma. In the absenceof dystrophin, mechanical stress leads to sarcolemmal ruptures, causingan uncontrolled influx of calcium into the muscle fiber interior,thereby triggering calcium-activated proteases and fiber necrosis.

For most genetic muscular dystrophies no clinically applicable andeffective therapies are currently available. Exon skipping techniquesare nowadays explored in order to combat genetic muscular dystrophies.Promising results have recently been reported by us and others on agenetic therapy aimed at restoring the reading frame of the dystrophinpre-mRNA in cells from the mdx mouse, the GRMD dog (reference 59) andDMD patients¹⁻¹¹. By the targeted skipping of a specific exon, a DMDphenotype (lacking dystrophin) is converted into a milder BMD phenotype(partly to largely functional dystrophin). The skipping of an exon ispreferably induced by the binding of antisense oligoribonucleotides(AONs) targeting either one or both of the splice sites, orexon-internal sequences. Since an exon will only be included in the mRNAwhen both the splice sites are recognised by the spliceosome complex,splice sites have been considered obvious targets for AONs. Morepreferably, one or more AONs are used which are specific for at leastpart of one or more exonic sequences involved in correct splicing of theexon. Using exon-internal AONs specific for an exon 46 sequence, we werepreviously able to modulate the splicing pattern in cultured myotubesfrom two different DMD patients with an exon 45 deletion¹¹. FollowingAON treatment, exon 46 was skipped, which resulted in a restored readingframe and the induction of dystrophin synthesis in at least 75% of thecells. We have recently shown that exon skipping can also efficiently beinduced in human control and patient muscle cells for 39 different DMDexons using exon-internal AONs^(1, 2, 11-15).

Hence, exon skipping techniques applied on the dystrophin gene result inthe generation of at least partially functional—albeitshorter—dystrophin protein in DMD patients. Since DMD is caused by adysfunctional dystrophin protein, it would be expected that the symptomsof DMD are sufficiently alleviated once a DMD patient has been providedwith functional dystrophin protein. However, the present inventionprovides the insight that, even though exon skipping techniques arecapable of inducing dystrophin synthesis, the oligonucleotide used forexon skipping technique can be improved any further by incorporating aninosine and/or a nucleotide containing a base able to form a wobble basepair in said oligonucleotide.

DESCRIPTION OF THE INVENTION

Oligonucleotide

In a first aspect, there is provided an oligonucleotide comprising aninosine and/or a nucleotide containing a base able to form a wobble basepair or a functional equivalent thereof, wherein the oligonucleotide, ora functional equivalent thereof, comprises a sequence which iscomplementary to at least part of a dystrophin pre-mRNA exon or at leastpart of a non-exon region of a dystrophin pre-mRNA said part being acontiguous stretch comprising at least 8 nucleotides.

The use of an inosine and/or a nucleotide containing a base able to forma wobble base pair in an oligonucleotide of the invention is veryattractive as explained below. Inosine for example is a known modifiedbase which can pair with three bases: uracil, adenine, and cytosine.Inosine is a nucleoside that is formed when hypoxanthine is attached toa ribose ring (also known as a ribofuranose) via a ß-N9-glycosidic bond.Inosine is commonly found in tRNAs and is essential for propertranslation of the genetic code in wobble base pairs. A wobble base pairis a G-U and I-U/I-A/I-C pair fundamental in RNA secondary structure.Its thermodynamic stability is comparable to that of the Watson-Crickbase pair. Wobble base pairs are critical for the proper translation ofthe genetic code. The genetic code makes up for disparities in thenumber of amino acids (20) for triplet codons (64), by using modifiedbase pairs in the first base of the anti-codon. Similarly, whendesigning primers for polymerase chain reaction, inosine is useful inthat it will indiscriminately pair with adenine, thymine, or cytosine. Afirst advantage of using such a base allows one to design a primer thatspans a single nucleotide polymorphism (SNP), without worry that thepolymorphism will disrupt the primer's annealing efficiency. Thereforein the invention, the use of such a base allows to design anoligonucleotide that may be used for an individual having a SNP withinthe dystrophin pre-mRNA stretch which is targeted by an oligonucleotideof the invention. A second advantage of using an inosine and/or a baseable to form a wobble base pair in an oligonucleotide of the inventionis when said oligonucleotide would normally contain a CpG if one wouldhave designed it as being complementary to at least part of a dystrophinpre-mRNA exon or at least part of a non-exon region of a dystrophinpre-mRNA said part being a contiguous stretch comprising at least 8nucleotides. The presence of a CpG in an oligonucleotide is usuallyassociated with an increased immunogenicity of said oligonucleotide(reference 60). This increased immunogenicity is undesired since it mayinduce the breakdown of muscle fibers. Replacing one, two or more CpG bythe corresponding inosine and/or a base able to form a wobble base pairin said oligonucleotide is expected to provide an oligonucleotide with adecreased and/or acceptable level of immunogenicity. Immunogenicity maybe assessed in an animal model by assessing the presence of CD4⁺ and/orCD8⁺ cells and/or inflammatory mononucleocyte infiltration in musclebiopsy of said animal.

Immunogenicity may also be assessed in blood of an animal or of a humanbeing treated with an oligonucleotide of the invention by detecting thepresence of a neutralizing antibody and/or an antibody recognizing saidoligonucleotide using a standard immunoassay known to the skilledperson.

An increase in immunogenicity preferably corresponds to a detectableincrease of at least one of these cell types by comparison to the amountof each cell type in a corresponding muscle biopsy of an animal beforetreatment or treated with a corresponding oligonucleotide having atleast one inosine and/or a base able to form a wobble base pair.Alternatively, an increase in immunogenicity may be assessed bydetecting the presence or an increasing amount of a neutralizingantibody or an antibody recognizing said oligonucleotide using astandard immunoassay.

A decrease in immunogenicity preferably corresponds to a detectabledecrease of at least one of these cell types by comparison to the amountof corresponding cell type in a corresponding muscle biopsy of an animalbefore treatment or treated with a corresponding oligonucleotide havingno inosine and/or a base able to form a wobble base pair. Alternativelya decrease in immunogenicity may be assessed by the absence of or adecreasing amount of said compound and/or neutralizing antibodies usinga standard immunoassay.

A third advantage of using an inosine and/or a base able to form awobble base pair in an oligonucleotide of the invention is to avoid ordecrease a potential multimerisation or aggregation of oligonucleotides.It is for example known that an oligonucleotide comprising a G-quartetmotif has the tendency to form a quadruplex, a multimer or aggregateformed by the Hoogsteen base-pairing of four single-strandedoligonucleotides (reference 61), which is of course not desired: as aresult the efficiency of the oligonucleotide is expected to bedecreased. Multimerisation or aggregation is preferably assessed bystandard polyacrylamid non-denaturing gel electrophoresis techniquesknown to the skilled person. In a preferred embodiment, less than 20% or15%, 10%, 7%, 5% or less of a total amount of an oligonucleotide of theinvention has the capacity to multimerise or aggregate assessed usingthe assay mentioned above.

A fourth advantage of using an inosine and/or a base able to form awobble base pair in an oligonucleotide of the invention is thus also toavoid quadruplex structures which have been associated withantithrombotic activity (reference 62) as well as with the binding to,and inhibition of, the macrophage scavenger receptor (reference 63).

A fifth advantage of using an inosine and/or a base able to form awobble base pair in an oligonucleotide of the invention is to allow todesign an oligonucleotide with improved RNA binding kinetics and/orthermodynamic properties. The RNA binding kinetics and/or thermodynamicproperties are at least in part determined by the melting temperature ofan oligonucleotide (Tm; calculated with the oligonucleotide propertiescalculator (http://www.unc.edu/˜cail/biotool/oligo/index.html) forsingle stranded RNA using the basic Tm and the nearest neighbour model),and/or the free energy of the AON-target exon complex (using RNAstructure version 4.5). If a Tm is too high, the oligonucleotide isexpected to be less specific. An acceptable Tm and free energy depend onthe sequence of the oligonucleotide. Therefore, it is difficult to givepreferred ranges for each of these parameters. An acceptable Tm may beranged between 35 and 65° C. and an acceptable free energy may be rangedbetween 15 and 45 kcal/mol.

The skilled person may therefore first choose an oligonucleotide as apotential therapeutic compound. In a second step, he may use theinvention to further optimise said oligonucleotide by decreasing itsimmunogenicity and/or avoiding aggregation and/or quadruplex formationand/or by optimizing its Tm and/or free energy of the AON-targetcomplex. He may try to introduce at least one inosine and/or a base ableto form a wobble base pair in said oligonucleotide at a suitableposition and assess how the immunogenicity and/or aggregation and/orquadruplex formation and/or Tm and/or free energy of the AON-targetcomplex have been altered by the presence of said inosine and/or a baseable to form a wobble base pair. If the alteration does not provide thedesired alteration or decrease of immunogenicity and/or aggregationand/or quadruplex formation and/or its Tm and/or free energy of theAON-target complex he may choose to introduce a further inosine and/or abase able to form a wobble base pair in said oligonucleotide and/or tointroduce a given inosine and/or a base able to form a wobble base pairat a distinct suitable position within said oligonucleotide.

An oligonucleotide comprising an inosine and/or a base able to form awobble base pair may be defined as an oligonucleotide wherein at leastone nucleotide has been substituted with an inosine and/or a base ableto form a wobble base pair. The skilled person knows how to test whethera nucleotide contains a base able to form a wobble base pair. Since forexample inosine can form a base pair with uracil, adenine, and/orcytosine, it means that at least one nucleotide able to form a base pairwith uracil, adenine and/or cytosine has been substituted with inosine.However, in order to safeguard specificity, the inosine containingoligonucleotide preferably comprises the substitution of at least one,two, three, four nucleotide(s) able to form a base pair with uracil oradenine or cytosine as long as an acceptable level of a functionalactivity of said oligonucleotide is retained as defined later herein.

An oligonucleotide comprising an inosine and/or a base able to form awobble base pair is preferably an olignucleotide, which is still able toexhibit an acceptable level of a functional activity of a correspondingoligonucleotide not comprising an inosine and/or a base able to form awobble base pair. A functional activity of said oligonucleotide ispreferably to provide an individual with a functional dystrophin proteinanchor mRNA and/or at least in part decreasing the production of anaberrant dystrophin protein anchor mRNA. Each of these features arelater defined herein. An acceptable level of such a functional activityis preferably at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of thefunctional activity of the corresponding oligonucleotide which does notcomprise an inosine and/or a base able to form a wobble base pair. Suchfunctional activity may be as measured in a muscular tissue or in amuscular cell of an individual or in vitro in a cell by comparison tothe functional activity of a corresponding oligonucleotides notcomprising an inosine and/or a base able to form a wobble base pair. Theassessment of the functionality may be carried out at the mRNA level,preferably using RT-PCR. The assessment of the functionality may becarried out at the protein level, preferably using western blot analysisor immunofluorescence analysis of cross-sections.

Within the context of the invention, an inosine and/or a base able toform a wobble base pair as present in an oligonucleotide is/are presentin a part of said oligonucleotide which is complementary to at leastpart of a dystrophin pre-mRNA exon or at least part of a non-exon regionof a dystrophin pre-mRNA said part being a contiguous stretch comprisingat least 8 nucleotides. Therefore, in a preferred embodiment, anoligonucleotide comprising an inosine and/or a nucleotide containing abase able to form a wobble base pair or a functional equivalent thereof,wherein the oligonucleotide, or a functional equivalent thereof,comprises a sequence which is complementary to at least part of adystrophin pre-mRNA exon or at least part of a non-exon region of adystrophin pre-mRNA said part being a contiguous stretch comprising atleast 8 nucleotides and wherein said inosine and/or a nucleotidecontaining a base able is/are present within the oligonucleotidesequence which is complementary to at least part of a dystrophinpre-mRNA as defined in previous sentence.

However, as later defined herein such inosine and/or a base able to forma wobble base pair may also be present in a linking moiety present in anoligonucleotide of the invention. Preferred linking moieties are laterdefined herein.

In a preferred embodiment, such oligonucleotide is preferably amedicament. More preferably, said medicament is for preventing ortreating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in anindividual or a patient. As defined herein a DMD pre-mRNA preferablymeans the pre-mRNA of a DMD gene of a DMD or BMD patient. A patient ispreferably intended to mean a patient having DMD or BMD or a patientsusceptible to develop DMD or BMD due to his or her genetic background.In the case of a DMD patient, an oligonucleotide used will preferablycorrect at least one of the DMD mutations as present in the DMD gene ofsaid patient and therefore will preferably create a dystrophin that willlook like a BMD dystrophin: said dystropin will preferably be afunctional dystrophin as later defined herein.

In the case of a BMD patient, an oligonucleotide as used will preferablycorrect at least one of the BMD mutations as present in the DMD gene ofsaid patient and therefore will preferably create a, or more of a,dystrophin, which will be more functional than the dystrophin which wasoriginally present in said BMD patient. Even more preferably, saidmedicament provides an individual with a functional or more (of a)functional dystrophin protein and/or mRNA and/or at least in partdecreases the production of an aberrant dystrophin protein and/or mRNA.

Preferably, a method of the invention by inducing and/or promotingskipping of at least one exon of the DMD pre-mRNA as identified hereinin one or more cells, preferably muscle cells of a patient, providessaid patient with an increased production of a more (of a) functionaldystrophin protein and/or mRNA and/or decreases the production of anaberrant or less functional dystrophin protein and/or mRNA in saidpatient.

Providing a patient with a more functional dystrophin protein and/ormRNA and/or decreasing the production of an aberrant dystrophin proteinand/or mRNA in said patient is typically applied in a DMD patient.Increasing the production of a more functional or functional dystrophinand/or mRNA is typically applied in a BMD patient.

Therefore a preferred method is a method, wherein a patient or one ormore cells of said patient is provided with an increased production of amore functional or functional dystrophin protein and/or mRNA and/orwherein the production of an aberrant dystrophin protein and/or mRNA insaid patient is decreased, wherein the level of said aberrant or morefunctional dystrophin protein and/or mRNA is assessed by comparison tothe level of said dystrophin and/or mRNA in said patient at the onset ofthe method.

As defined herein, a functional dystrophin is preferably a wild typedystrophin corresponding to a protein having the amino acid sequence asidentified in SEQ ID NO: 1. A functional dystrophin is preferably adystrophin, which has an actin binding domain in its N terminal part(first 240 amino acids at the N terminus), a cystein-rich domain (aminoacid 3361 till 3685) and a C terminal domain (last 325 amino acids atthe C terminus) each of these domains being present in a wild typedystrophin as known to the skilled person. The amino acids indicatedherein correspond to amino acids of the wild type dystrophin beingrepresented by SEQ ID NO: 1. In another embodiment, a functionaldystrophin is a dystrophin, which exhibits at least to some extent anactivity of a wild type dystrophin. “At least to some extent” preferablymeans at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of acorresponding activity of a wild type functional dystrophin. In thiscontext, an activity of a wild type dystrophin is preferably binding toactin and to the dystrophin-associated glycoprotein complex (DGC)⁵⁶.Binding of dystrophin to actin and to the DGC complex may be visualizedby either co-immunoprecipitation using total protein extracts orimmunofluorescence analysis of cross-sections, from a biopsy of a musclesuspected to be dystrophic, as known to the skilled person.

Individuals suffering from Duchenne muscular dystrophy typically have amutation in the gene encoding dystrophin that prevents synthesis of thecomplete protein, i.e a premature stop prevents the synthesis of theC-terminus of the protein. In Becker muscular dystrophy the dystrophingene also comprises a mutation compared to the wild type but themutation does typically not include a premature stop and the C-terminusof the protein is typically synthesized. As a result a functionaldystrophin protein is synthesized that has at least the same activity inkind as a wild type protein, although not necessarily the same amount ofactivity. In a preferred embodiment, a functional dystrophin proteinmeans an in frame dystrophin gene. The genome of a BMD individualtypically encodes a dystrophin protein comprising the N terminal part(first 240 amino acids at the N terminus), a cystein-rich domain (aminoacid 3361 till 3685) and a C terminal domain (last 325 amino acids atthe C terminus) but its central rod shaped domain may be shorter thanthe one of a wild type dystrophin⁵⁶. Exon-skipping for the treatment ofDMD is preferably but not exclusively directed to overcome a prematurestop in the pre-mRNA by skipping an exon in the rod-domain shaped domainto correct the reading frame and allow synthesis of remainder of thedystrophin protein including the C-terminus, albeit that the protein issomewhat smaller as a result of a smaller rod domain. In a preferredembodiment, an individual having DMD and being treated using anoligonucleotide as defined herein will be provided a dystrophin, whichexhibits at least to some extent an activity of a wild type dystrophin.More preferably, if said individual is a Duchenne patient or issuspected to be a Duchenne patient, a functional dystrophin is adystrophin of an individual having BMD: preferably said dystrophin isable to interact with both actin and the DGC, but its central rod shapeddomain may be shorter than the one of a wild type dystrophin(Aartsma-Rus et al (2006, ref 56). The central rod domain of wild typedystrophin comprises 24 spectrin-like repeats⁵⁶. For example, a centralrod shaped domain of a dystrophin as provided herein may comprise 5 to23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind toactin and to DGC. Decreasing the production of an aberrant dystrophin insaid patient or in a cell of said patient may be assessed at the mRNAlevel and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA,is still detectable by RT PCR. An aberrant dystrophin mRNA or protein isalso referred to herein as a non-functional or less to non-functional orsemi-functional dystrophin mRNA or protein. A non-functional pre-mRNAdystrophin is preferably leads to an out of frame dystrophin protein,which means that no dystrophin protein will be produced and/or detected.A non functional dystrophin protein is preferably a dystrophin proteinwhich is not able to bind actin and/or members of the DGC proteincomplex. A non-functional dystrophin protein or dystrophin mRNA doestypically not have, or does not encode a dystrophin protein with anintact C-terminus of the protein.

Increasing the production of a functional dystrophin in said patient orin a cell of said patient may be assessed at the mRNA level (by RT-PCRanalysis) and preferably means that a detectable amount of a functionalor in frame dystrophin mRNA is detectable by RT PCR. In anotherembodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or moreof the detectable dystrophin mRNA is a functional or in frame dystrophinmRNA.

Increasing the production of a functional dystrophin in said patient orin a cell of said patient may be assessed at the protein level (byimmunofluorescence and western blot analyses) and preferably means thata detectable amount of a functional dystrophin protein is detectable byimmunofluorescence or western blot analysis. In another embodiment, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of thedetectable dystrophin protein is a functional dystrophin protein.

An increase or a decrease is preferably assessed in a muscular tissue orin a muscular cell of an individual or a patient by comparison to theamount present in said individual or patient before treatment with saidmolecule or composition of the invention. Alternatively, the comparisoncan be made with a muscular tissue or cell of said individual orpatient, which has not yet been treated with said oligonucleotide orcomposition in case the treatment is local.

In a preferred method, one or more symptom(s) from a DMD or a BMDpatient is/are alleviated and/or one or more characteristic(s) of amuscle cell or tissue from a DMD or a BMD patient is/are alleviatedusing a molecule or a composition of the invention. Such symptoms may beassessed on the patient self. Such characteristics may be assessed atthe cellular, tissue level of a given patient. An alleviation of one ormore characteristics may be assessed by any of the following assays on amyogenic cell or muscle cell from a patient: reduced calcium uptake bymuscle cells, decreased collagen synthesis, altered morphology, alteredlipid biosynthesis, decreased oxidative stress, and/or improved musclefiber function, integrity, and/or survival. These parameters are usuallyassessed using immunofluorescence and/or histochemical analyses of crosssections of muscle biopsies.

Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy orBecker Muscular Dystrophy in an individual using a molecule or acomposition of the invention may be assessed by any of the followingassays: prolongation of time to loss of walking, improvement of musclestrength, improvement of the ability to lift weight, improvement of thetime taken to rise from the floor, improvement in the nine-meter walkingtime, improvement in the time taken for four-stairs climbing,improvement of the leg function grade, improvement of the pulmonaryfunction, improvement of cardiac function, improvement of the quality oflife. Each of these assays is known to the skilled person. As anexample, the publication of Manzur at al (2008, ref 58) gives anextensive explanation of each of these assays. For each of these assays,as soon as a detectable improvement or prolongation of a parametermeasured in an assay has been found, it will preferably mean that one ormore symptoms of Duchenne Muscular Dystrophy or Becker MuscularDystrophy has been alleviated in an individual using a molecule orcomposition of the invention. Detectable improvement or prolongation ispreferably a statistically significant improvement or prolongation asdescribed in Hodgetts et al (2006, ref 57). Alternatively, thealleviation of one or more symptom(s) of Duchenne Muscular Dystrophy orBecker Muscular Dystrophy may be assessed by measuring an improvement ofa muscle fiber function, integrity and/or survival as later definedherein.

An oligonucleotide as used herein preferably comprises an antisenseoligonucleotide or antisense oligoribonucleotide. In a preferredembodiment an exon skipping technique is applied. Exon skippinginterferes with the natural splicing processes occurring within aeukaryotic cell. In higher eukaryotes the genetic information forproteins in the DNA of the cell is encoded in exons which are separatedfrom each other by intronic sequences. These introns are in some casesvery long. The transcription machinery of eukaryotes generates apre-mRNA which contains both exons and introns, while the splicingmachinery, often already during the production of the pre-mRNA,generates the actual coding region for the protein by splicing togetherthe exons present in the pre-mRNA.

Exon-skipping results in mature mRNA that lacks at least one skippedexon. Thus, when said exon codes for amino acids, exon skipping leads tothe expression of an altered product. Technology for exon-skipping iscurrently directed towards the use of antisense oligonucleotides (AONs).Much of this work is done in the mdx mouse model for Duchenne musculardystrophy. The mdx mouse carries a nonsense mutation in exon 23. Despitethe mdx mutation, which should preclude the synthesis of a functionaldystrophin protein, rare, naturally occurring dystrophin positive fibershave been observed in mdx muscle tissue. These dystrophin-positivefibers are thought to have arisen from an apparently naturally occurringexon-skipping mechanism, either due to somatic mutations or throughalternative splicing. AONs directed to, respectively, the 3′ and/or 5′splice sites of introns 22 and 23 in dystrophin pre-mRNA, have beenshown to interfere with factors normally involved in removal of intron23 so that also exon 23 was removed from the mRNA^(3, 5, 6, 89, 40).

By the targeted skipping of a specific exon, a DMD phenotype isconverted into a milder BMD phenotype. The skipping of an exon ispreferably induced by the binding of AONs targeting either one or bothof the splice sites, or exon-internal sequences. An oligonucleotidedirected toward an exon internal sequence typically exhibits no overlapwith non-exon sequences. It preferably does not overlap with the splicesites at least not insofar, as these are present in the intron. Anoligonucleotide directed toward an exon internal sequence preferablydoes not contain a sequence complementary to an adjacent intron. Furtherprovided is thus an oligonucleotide according to the invention, whereinsaid oligonucleotide, or a functional equivalent thereof, is forinhibiting inclusion of an exon of a dystrophin pre-mRNA into mRNAproduced from splicing of said pre-mRNA. An exon skipping technique ispreferably applied such that the absence of an exon from mRNA producedfrom dystrophin pre-mRNA generates a coding region for a morefunctional—albeit shorter—dystrophin protein. In this context,inhibiting inclusion of an exon preferably means that the detection ofthe original, aberrant dystrophin mRNA and/or protein is decreased asearlier defined herein.

Since an exon of a dystrophin pre-mRNA will only be included into theresulting mRNA when both the splice sites are recognised by thespliceosome complex, splice sites have been obvious targets for AONs.One embodiment therefore provides an oligonucleotide, or a functionalequivalent thereof, comprising a sequence which is complementary to anon-exon region of a dystrophin pre mRNA. In one embodiment an AON isused which is solely complementary to a non-exon region of a dystrophinpre mRNA. This is however not necessary: it is also possible to use anAON which comprises an intron-specific sequence as well as exon-specificsequence. Such AON comprises a sequence which is complementary to anon-exon region of a dystrophin pre mRNA, as well as a sequence which iscomplementary to an exon region of a dystrophin pre mRNA. Of course, anAON is not necessarily complementary to the entire sequence of adystrophin exon or intron. AONs, which are complementary to a part ofsuch exon or intron are preferred. An AON is preferably complementary toat least part of a dystrophin exon and/or intron, said part having atleast 8, 10, 13, 15, 20 nucleotides.

Splicing of a dystrophin pre-mRNA occurs via two sequentialtransesterification reactions. First, the 2′OH of a specificbranch-point nucleotide within the intron that is defined duringspliceosome assembly performs a nucleophilic attack on the firstnucleotide of the intron at the 5′ splice site forming the lariatintermediate. Second, the 3′OH of the released 5′ exon then performs anucleophilic attack at the last nucleotide of the intron at the 3′splice site thus joining the exons and releasing the intron lariat. Thebranch point and splice sites of an intron are thus involved in asplicing event. Hence, an oligonucleotide comprising a sequence, whichis complementary to such branch point and/or splice site is preferablyused for exon skipping. Further provided is therefore anoligonucleotide, or a functional equivalent thereof, which comprises asequence which is complementary to a splice site and/or branch point ofa dystrophin pre mRNA.

Since splice sites contain consensus sequences, the use of anoligonucleotide or a functional equivalent thereof (herein also calledan AON) comprising a sequence which is complementary of a splice siteinvolves the risk of promiscuous hybridization. Hybridization of AONs toother splice sites than the sites of the exon to be skipped could easilyinterfere with the accuracy of the splicing process. To overcome theseand other potential problems related to the use of AONs which arecomplementary to an intron sequence, one preferred embodiment providesan oligonucleotide, or a functional equivalent thereof, comprising asequence which is complementary to a dystrophin pre-mRNA exon.Preferably, said AON is capable of specifically inhibiting an exoninclusion signal of at least one exon in said dystrophin pre-mRNA.Interfering with an exon inclusion signal (EIS) has the advantage thatsuch elements are located within the exon. By providing an AON for theinterior of the exon to be skipped, it is possible to interfere with theexon inclusion signal thereby effectively masking the exon from thesplicing apparatus. The failure of the splicing apparatus to recognizethe exon to be skipped thus leads to exclusion of the exon from thefinal mRNA. This embodiment does not interfere directly with theenzymatic process of the splicing machinery (the joining of the exons).It is thought that this allows the method to be more specific and/orreliable. It is thought that an EIS is a particular structure of an exonthat allows splice acceptor and donor to assume a particular spatialconformation. In this concept, it is the particular spatial conformationthat enables the splicing machinery to recognize the exon. However, theinvention is certainly not limited to this model. In a preferredembodiment, use is made of an oligonucleotide, which is capable ofbinding to an exon and is capable of inhibiting an EIS. An AON mayspecifically contact said exon at any point and still be able tospecifically inhibit said EIS.

Within the context of the invention, a functional equivalent of anoligonucleotide preferably means an oligonucleotide as defined hereinwherein one or more nucleotides have been substituted and wherein anactivity of said functional equivalent is retained to at least someextent. Preferably, an activity of said functional equivalent isproviding a functional dystrophin protein. Said activity of saidfunctional equivalent is therefore preferably assessed by quantifyingthe amount of a functional dystrophin protein or by quantifying theamount of a functional dystrophin mRNA. A functional dystrophin protein(or a functional dystrophin mRNA) is herein preferably defined as beinga dystrophin protein (or a dystrophin protein encoded by said mRNA) ableto bind actin and members of the DGC protein. The assessment of saidactivity of an oligonucleotide is preferably done by RT-PCR (m-RNA) orby immunofluorescence or Western blot analyses (protein). Said activityis preferably retained to at least some extent when it represents atleast 50%, or at least 60%, or at least 70% or at least 80% or at least90% or at least 95% or more of corresponding activity of saidoligonucleotide the functional equivalent derives from. Such activitymay be measured in a muscular tissue or in a muscular cell of anindividual or in vitro in a cell by comparison to an activity of acorresponding oligonucleotide of said oligonucleotide the functionalequivalent derives from. Throughout this application, when the wordoligonucleotide is used it may be replaced by a functional equivalentthereof as defined herein.

Hence, an oligonucleotide, or a functional equivalent thereof,comprising or consisting of a sequence which is complementary to adystrophin pre-mRNA exon provides good DMD therapeutic results. In onepreferred embodiment an oligonucleotide, or a functional equivalentthereof, is used which comprises or consists of a sequence which iscomplementary to at least part of either dystrophin pre-mRNA exons 2 to75 said part having or comprising at least 13 nucleotides. However, saidpart may also have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Apart of dystrophin pre-mRNA to which an oligonucleotide is complementarymay also be called a contiguous stretch of dystrophin pre-mRNA.

Most preferably an AON is used which comprises or consists of a sequencewhich is complementary to at least part of dystrophin pre-mRNA exon 51,45, 53, 44, 46, 52, 50, 43, 6, 7, 8, 55, 2, 11, 17, 19, 21, 57, 59, 62,63, 65, 66, 69, and/or 75 said part having or comprising at least 13nucleotides. However, said part may also have at least 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50 nucleotides. More preferred oligonucleotides are represented by asequence that comprises or consists of each of the following sequencesSEQ ID NO: 2 to SEQ ID NO:539 wherein at least one inosine and/or a baseable to form a wobble base pair is present in said sequence. Preferably,an inosine has been introduced in one of these sequences to replace aguanosine, adenine, or a uracil. Accordingly, an even more preferredoligonucleotide as used herein is represented by a sequence thatcomprises or consists of SEQ ID NO:2 to SEQ ID NO:486 or SEQ ID NO:539,even more preferably SEQ ID NO:2 to NO 237 or SEQ ID NO:539, mostpreferably SEQ ID NO:76 wherein at least one inosine and/or a base ableto form a wobble base pair is present in said sequence. Preferably, aninosine has been introduced in one of these sequences to replace aguanosine, adenine, or a uracil.

Accordingly, in another preferred embodiment, an oligonucleotide as usedherein is represented by a sequence that comprises or consists of SEQ IDNO:540 to SEQ ID NO:576. More preferably, an oligonucleotide as usedherein is represented by a sequence that comprises or consists of SEQ IDNO:557.

Said exons are listed in decreasing order of patient populationapplicability. Hence, the use of an AON comprising a sequence, which iscomplementary to at least part of dystrophin pre-mRNA exon 51 issuitable for use in a larger part of the DMD patient population ascompared to an AON comprising a sequence which is complementary todystrophin pre-mRNA exon 44, et cetera.

In a preferred embodiment, an oligonucleotide of the invention, whichcomprises a sequence that is complementary to part of dystrophinpre-mRNA is such that the complementary part is at least 50% of thelength of the oligonucleotide of the invention, more preferably at least60%, even more preferably at least 70%, even more preferably at least80%, even more preferably at least 90% or even more preferably at least95%, or even more preferably 98% or even more preferably at least 99%,or even more preferably 100%. In a most preferred embodiment, theoligonucleotide of the invention consists of a sequence that iscomplementary to part of dystrophin pre-mRNA as defined herein. As anexample, an oligonucleotide may comprise a sequence that iscomplementary to part of dystrophin pre-mRNA as defined herein andadditional flanking sequences. In a more preferred embodiment, thelength of said complementary part of said oligonucleotide is of at least8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 nucleotides. Preferably, additional flankingsequences are used to modify the binding of a protein to theoligonucleotide, or to modify a thermodynamic property of theoligonucleotide, more preferably to modify target RNA binding affinity.

One preferred embodiment provides an oligonucleotide, or a functionalequivalent thereof which comprises:

a sequence which is complementary to a region of a dystrophin pre-mRNAexon that is hybridized to another part of a dystrophin pre-mRNA exon(closed structure), and

a sequence which is complementary to a region of a dystrophin pre-mRNAexon that is not hybridized in said dystrophin pre-mRNA (openstructure).

For this embodiment, reference is made to WO 2004/083432, which isincorporated by reference in its entirety. RNA molecules exhibit strongsecondary structures, mostly due to base pairing of complementary orpartly complementary stretches within the same RNA. It has long sincebeen thought that structures in the RNA play a role in the function ofthe RNA. Without being bound by theory, it is believed that thesecondary structure of the RNA of an exon plays a role in structuringthe splicing process. The structure of an exon is one parameter which isbelieved to direct its inclusion into the mRNA. However, otherparameters may also play a role therein. Herein this signalling functionis referred to as an exon inclusion signal. A complementaryoligonucleotide of this embodiment is capable of interfering with thestructure of the exon and thereby capable of interfering with the exoninclusion signal of the exon. It has been found that many complementaryoligonucleotides indeed comprise this capacity, some more efficient thanothers. Oligonucleotides of this preferred embodiment, i.e. those withthe said overlap directed towards open and closed structures in thenative exon RNA, are a selection from all possible oligonucleotides. Theselection encompasses oligonucleotides that can efficiently interferewith an exon inclusion signal. Without being bound by theory it isthought that the overlap with an open structure improves the invasionefficiency of the oligonucleotide and prevents the binding of splicingfactors (i.e. increases the efficiency with which the oligonucleotidecan enter the structure), whereas the overlap with the closed structuresubsequently increases the efficiency of interfering with the secondarystructure of the RNA of the exon, and thereby interfere with the exoninclusion signal. It is found that the length of the partialcomplementarity to both the closed and the open structure is notextremely restricted. We have observed high efficiencies witholigonucleotides with variable lengths of complementarity in eitherstructure. The term complementarity is used herein to refer to a stretchof nucleic acids that can hybridise to another stretch of nucleic acidsunder physiological conditions. It is thus not absolutely required thatall the bases in the region of complementarity are capable of pairingwith bases in the opposing strand. For instance, when designing theoligonucleotide one may want to incorporate for instance a residue thatdoes not base pair with the base on the complementary strand. Mismatchesmay, to some extent, be allowed, if under the circumstances in the cell,the stretch of nucleotides is sufficiently capable of hybridising to thecomplementary part. In this context, “sufficiently” preferably meansthat using a gel mobility shift assay as described in example 1 of EP 1619 249, binding of an oligonucleotide is detectable. Optionally, saidoligonucleotide may further be tested by transfection into muscle cellsof patients. Skipping of the targeted exon may be assessed by RT-PCR (asdescribed in EP 1 619 249). The complementary regions are preferablydesigned such that, when combined, they are specific for the exon in thepre-mRNA. Such specificity may be created with various lengths ofcomplementary regions as this depends on the actual sequences in other(pre-)mRNA in the system. The risk that also one or more other pre-mRNAwill be able to hybridise to the oligonucleotide decreases withincreasing size of the oligonucleotide. It is clear thatoligonucleotides comprising mismatches in the region of complementaritybut that retain the capacity to hybridise to the targeted region(s) inthe pre-mRNA, can be used in the present invention. However, preferablyat least the complementary parts do not comprise such mismatches asthese typically have a higher efficiency and a higher specificity, thanoligonucleotides having such mismatches in one or more complementaryregions. It is thought, that higher hybridisation strengths, (i.e.increasing number of interactions with the opposing strand) arefavourable in increasing the efficiency of the process of interferingwith the splicing machinery of the system. Preferably, thecomplementarity is between 90 and 100%. In general this allows forapproximately 1 or 2 mismatch(es) in an oligonucleotide of around 20nucleotides

The secondary structure is best analysed in the context of the pre-mRNAwherein the exon resides. Such structure may be analysed in the actualRNA. However, it is currently possible to predict the secondarystructure of an RNA molecule (at lowest energy costs) quite well usingstructure-modelling programs. A non-limiting example of a suitableprogram is RNA mfold version 3.1 server⁴¹. A person skilled in the artwill be able to predict, with suitable reproducibility, a likelystructure of the exon, given the nucleotide sequence. Best predictionsare obtained when providing such modelling programs with both the exonand flanking intron sequences. It is typically not necessary to modelthe structure of the entire pre-mRNA.

The open and closed structure to which the oligonucleotide is directed,are preferably adjacent to one another. It is thought, that in this waythe annealing of the oligonucleotide to the open structure inducesopening of the closed structure whereupon annealing progresses into thisclosed structure. Through this action the previously closed structureassumes a different conformation. The different conformation results inthe disruption of the exon inclusion signal. However, when potential(cryptic) splice acceptor and/or donor sequences are present within thetargeted exon, occasionally a new exon inclusion signal is generateddefining a different (neo) exon, i.e. with a different 5′ end, adifferent 3′ end, or both. This type of activity is within the scope ofthe present invention as the targeted exon is excluded from the mRNA.The presence of a new exon, containing part of the targeted exon, in themRNA does not alter the fact that the targeted exon, as such, isexcluded. The inclusion of a neo-exon can be seen as a side effect,which occurs only occasionally. There are two possibilities when exonskipping is used to restore (part of) an open reading frame ofdystrophin that is disrupted as a result of a mutation. One is that theneo-exon is functional in the restoration of the reading frame, whereasin the other case the reading frame is not restored. When selectingoligonucleotides for restoring dystrophin reading frames by means ofexon-skipping it is of course clear that under these conditions onlythose oligonucleotides are selected that indeed result in exon-skippingthat restores the dystrophin open reading frame, with or without aneo-exon.

Further provided is an oligonucleotide, or a functional equivalentthereof, comprising a sequence that is complementary to a binding sitefor a serine-arginine (SR) protein in RNA of an exon of a dystrophinpre-mRNA. In WO 2006/112705 we have disclosed the presence of acorrelation between the effectivity of an exon-internal antisenseoligonucleotide (AON) in inducing exon skipping and the presence of a(for example by ESE finder) predicted SR binding site in the targetpre-mRNA site of said AON. Therefore, in one embodiment anoligonucleotide is generated comprising determining a (putative) bindingsite for an SR (Ser-Arg) protein in RNA of a dystrophin exon andproducing an oligonucleotide that is complementary to said RNA and thatat least partly overlaps said (putative) binding site. The term “atleast partly overlaps” is defined herein as to comprise an overlap ofonly a single nucleotide of an SR binding site as well as multiplenucleotides of said binding site as well as a complete overlap of saidbinding site. This embodiment preferably further comprises determiningfrom a secondary structure of said RNA, a region that is hybridised toanother part of said RNA (closed structure) and a region that is nothybridised in said structure (open structure), and subsequentlygenerating an oligonucleotide that at least partly overlaps said(putative) binding site and that overlaps at least part of said closedstructure and overlaps at least part of said open structure. In this waywe increase the chance of obtaining an oligonucleotide that is capableof interfering with the exon inclusion from the pre-mRNA into mRNA. Itis possible that a first selected SR-binding region does not have therequested open-closed structure in which case another (second) SRprotein binding site is selected which is then subsequently tested forthe presence of an open-closed structure. This process is continueduntil a sequence is identified which contains an SR protein binding siteas well as a(n) (partly overlapping) open-closed structure. Thissequence is then used to design an oligonucleotide which iscomplementary to said sequence.

Such a method, for generating an oligonucleotide, is also performed byreversing the described order, i.e. first generating an oligonucleotidecomprising determining, from a secondary structure of RNA from adystrophin exon, a region that assumes a structure that is hybridised toanother part of said RNA (closed structure) and a region that is nothybridised in said structure (open structure), and subsequentlygenerating an oligonucleotide, of which at least a part of saidoligonucleotide is complementary to said closed structure and of whichat least another part of said oligonucleotide is complementary to saidopen structure. This is then followed by determining whether an SRprotein binding site at least overlaps with said open/closed structure.In this way the method of WO 2004/083432 is improved. In yet anotherembodiment the selections are performed simultaneously.

Without wishing to be bound by any theory it is currently thought thatuse of an oligonucleotide directed to an SR protein binding site resultsin (at least partly) impairing the binding of an SR protein to thebinding site of an SR protein which results in disrupted or impairedsplicing.

Preferably, an open/closed structure and an SR protein binding sitepartly overlap and even more preferred an open/closed structurecompletely overlaps an SR protein binding site or an SR protein bindingsite completely overlaps an open/closed structure. This allows for animproved disruption of exon inclusion.

Besides consensus splice sites sequences, many (if not all) exonscontain splicing regulatory sequences such as exonic splicing enhancer(ESE) sequences to facilitate the recognition of genuine splice sites bythe spliceosome^(42, 43). A subgroup of splicing factors, called the SRproteins, can bind to these ESEs and recruit other splicing factors,such as U1 and U2AF to (weakly defined) splice sites. The binding sitesof the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55)have been analyzed in detail and these results are implemented in ESEfinder, a web source that predicts potential binding sites for these SRproteins^(42, 43) There is a correlation between the effectiveness of anAON and the presence/absence of an SF2/ASF, SC35 and SRp40 binding site.In a preferred embodiment, the invention thus provides a combination asdescribed above, wherein said SR protein is SF2/ASF or SC35 or SRp40.

In one embodiment an oligonucleotide, or a functional equivalent thereofis capable of specifically binding a regulatory RNA sequence which isrequired for the correct splicing of a dystrophin exon in a transcript.Several cis-acting RNA sequences are required for the correct splicingof exons in a transcript. In particular, supplementary elements such asintronic or exonic splicing enhancers (ISEs and ESEs) or silencers (ISSsand ESEs) are identified to regulate specific and efficient splicing ofconstitutive and alternative exons. Using sequence-specific antisenseoligonucleotides (AONs) that bind to the elements, their regulatoryfunction is disturbed so that the exon is skipped, as shown for DMD.Hence, in one preferred embodiment an oligonucleotide or functionalequivalent thereof is used which is complementary to an intronicsplicing enhancer (ISE), an exonic splicing enhancer (ESE), an intronicsplicing silencer (ISS) and/or an exonic splicing silencer (ESS). Asalready described herein before, a dystrophin exon is in one preferredembodiment skipped by an agent capable of specifically inhibiting anexon inclusion signal of said exon, so that said exon is not recognizedby the splicing machinery as a part that needs to be included in themRNA. As a result, a mRNA without said exon is formed.

An AON used herein is preferably complementary to a consecutive part ora contiguous stretch of between 8 and 50 nucleotides of dystrophin exonRNA or dystrophin intron RNA. In one embodiment an AON used herein iscomplementary to a consecutive part or a contiguous stretch of between14 and 50 nucleotides of a dystrophin exon RNA or dystrophin intron RNA.Preferably, said AON is complementary to a consecutive part orcontiguous stretch of between 14 and 25 nucleotides of said exon RNA.More preferably, an AON is used which comprises a sequence which iscomplementary to a consecutive part or a contiguous stretch of between20 and 25 nucleotides of a dystrophin exon RNA or a dystrophin intronRNA.

Different types of nucleic acid may be used to generate anoligonucleotide. Preferably, said oligonucleotide comprises RNA, asRNA/RNA hybrids are very stable. Since one of the aims of the exonskipping technique is to direct splicing in subjects it is preferredthat the oligonucleotide RNA comprises a modification providing the RNAwith an additional property, for instance resistance to endonucleases,exonucleases, and RNaseH, additional hybridisation strength, increasedstability (for instance in a bodily fluid), increased or decreasedflexibility, reduced toxicity, increased intracellular transport,tissue-specificity, etc. Preferably, said modification comprises a2′-O-methyl-phosphorothioate oligoribonucleotide modification.Preferably, said modification comprises a 2′-O-methyl-phosphorothioateoligo deoxyribonucleotide modification. One embodiment thus provides anoligonucleotide is used which comprises RNA which contains amodification, preferably a 2′-O-methyl modified ribose (RNA) ordeoxyribose (DNA) modification.

In one embodiment the invention provides a hybrid oligonucleotidecomprising an oligonucleotide comprising a 2′-O-methyl-phosphorothioateoligo(deoxy)ribonucleotide modification and locked nucleic acid. Thisparticular oligonucleotide comprises better sequence specificitycompared to an equivalent consisting of locked nucleic acid, andcomprises improved effectivity when compared with an oligonucleotideconsisting of 2′-O-methyl-phosphorothioate oligo(deoxy)ribonucleotidemodification.

With the advent of nucleic acid mimicking technology it has becomepossible to generate molecules that have a similar, preferably the samehybridisation characteristics in kind not necessarily in amount asnucleic acid itself. Such functional equivalents are of course alsosuitable for use in the invention. Preferred examples of functionalequivalents of an oligonucleotide are peptide nucleic acid and/or lockednucleic acid. Most preferably, a morpholino phosphorodiamidate is used.Suitable but non-limiting examples of equivalents of oligonucleotides ofthe invention can be found in⁴⁴⁻⁵⁰. Hybrids between one or more of theequivalents among each other and/or together with nucleic acid are ofcourse also suitable. In a preferred embodiment locked nucleic acid isused as a functional equivalent of an oligonucleotide, as locked nucleicacid displays a higher target affinity and reduced toxicity andtherefore shows a higher efficiency of exon skipping.

In one embodiment an oligonucleotide, or a functional equivalentthereof, which is capable of inhibiting inclusion of a dystrophin exoninto dystrophin mRNA is combined with at least one otheroligonucleotide, or functional equivalent thereof, that is capable ofinhibiting inclusion of another dystrophin exon into dystrophin mRNA.This way, inclusion of two or more exons of a dystrophin pre-mRNA inmRNA produced from this pre-mRNA is prevented. This embodiment isfurther referred to as double- or multi-exon skipping^(2, 15). In mostcases double-exon skipping results in the exclusion of only the twotargeted exons from the dystrophin pre-mRNA. However, in other cases itwas found that the targeted exons and the entire region in between saidexons in said pre-mRNA were not present in the produced mRNA even whenother exons (intervening exons) were present in such region. Thismulti-exon skipping was notably so for the combination ofoligonucleotides derived from the DMD gene, wherein one oligonucleotidefor exon 45 and one oligonucleotide for exon 51 was added to a celltranscribing the DMD gene. Such a set-up resulted in mRNA being producedthat did not contain exons 45 to 51. Apparently, the structure of thepre-mRNA in the presence of the mentioned oligonucleotides was such thatthe splicing machinery was stimulated to connect exons 44 and 52 to eachother. Other preferred examples of multi-exon skipping are:

-   -   the use of an oligonucleotide targeting exon 17, and a second        one exon 48 which may result in the skipping of said both exons        or of the entire region between exon 17 and exon 48.    -   the use of an oligonucleotide targeting exon 17, and a second        one exon 51 which may result in the skipping of said both exons        or of the entire region between exon 17 and exon 51.    -   the use of an oligonucleotide targeting exon 42, and a second        one exon 55 which may result in the skipping of said both exons        or of the entire region between exon 42 and exon 55.    -   the use of an oligonucleotide targeting exon 43, and a second        one exon 51 which may result in the skipping of said both exons        or of the entire region between exon 43 and exon 51.    -   the use of an oligonucleotide targeting exon 43, and a second        one exon 55 which may result in the skipping of said both exons        or of the entire region between exon 43 and exon 55.    -   the use of an oligonucleotide targeting exon 45, and a second        one exon 55 which may result in the skipping of said both exons        or of the entire region between exon 45 and exon 55.    -   the use of an oligonucleotide targeting exon 45, and a second        one exon 59 which may result in the skipping of said both exons        or of the entire region between exon 45 and exon 59.    -   the use of an oligonucleotide targeting exon 48, and a second        one exon 59 which may result in the skipping of said both exons        or of the entire region between exon 48 and exon 59.    -   the use of an oligonucleotide targeting exon 50, and a second        one exon 51 which may result in the skipping of said both exons.    -   the use of an oligonucleotide targeting exon 51, and a second        one exon 52 which may result in the skipping of said both exons.

Further provided is therefore an oligonucleotide which comprises atleast 8, preferably between 16 to 80, consecutive nucleotides that arecomplementary to a first exon of a dystrophin pre-mRNA and wherein anucleotide sequence is used which comprises at least 8, preferablybetween 16 to 80, consecutive nucleotides that are complementary to asecond exon of said dystrophin pre-mRNA. Said first and said second exonmay be the same.

In one preferred embodiment said first and said second exon areseparated in said dystrophin pre-mRNA by at least one exon to which saidoligonucleotide is not complementary. Alternatively, said first and saidsecond exon are adjacent.

It is possible to specifically promote the skipping of also theintervening exons by providing a linkage between the two complementaryoligonucleotides. Hence, in one embodiment stretches of nucleotidescomplementary to at least two dystrophin exons are separated by alinking moiety. The at least two stretches of nucleotides are thuslinked in this embodiment so as to form a single molecule. Furtherprovided is therefore an oligonucleotide, or functional equivalentthereof which is complementary to at least two exons in a dystrophinpre-mRNA, said oligonucleotide or functional equivalent comprising atleast two parts wherein a first part comprises an oligonucleotide havingat least 8, preferably between 16 to 80, consecutive nucleotides thatare complementary to a first of said at least two exons and wherein asecond part comprises an oligonucleotide having at least 8, preferablybetween 16 to 80, consecutive nucleotides that are complementary to asecond exon in said dystrophin pre-mRNA. The linkage may be through anymeans, but is preferably accomplished through a nucleotide linkage. Inthe latter case, the number of nucleotides that do not contain anoverlap between one or the other complementary exon can be zero, but ispreferably between 4 to 40 nucleotides. The linking moiety can be anytype of moiety capable of linking oligonucleotides. Preferably, saidlinking moiety comprises at least 4 uracil nucleotides. Currently, manydifferent compounds are available that mimic hybridisationcharacteristics of oligonucleotides. Such a compound, called herein afunctional equivalent of an oligonucleotide, is also suitable for thepresent invention if such equivalent comprises similar hybridisationcharacteristics in kind not necessarily in amount. Suitable functionalequivalents are mentioned earlier in this description. As mentioned,oligonucleotides of the invention do not have to consist of onlyoligonucleotides that contribute to hybridisation to the targeted exon.There may be additional material and/or nucleotides added.

The DMD gene is a large gene, with many different exons. Consideringthat the gene is located on the X-chromosome, it is mostly boys that areaffected, although girls can also be affected by the disease, as theymay receive a bad copy of the gene from both parents, or are sufferingfrom a particularly biased inactivation of the functional allele due toa particularly biased X chromosome inactivation in their muscle cells.The protein is encoded by a plurality of exons (79) over a range of atleast 2.4 Mb. Defects may occur in any part of the DMD gene. Skipping ofa particular exon or particular exons can, very often, result in arestructured mRNA that encodes a shorter than normal but at leastpartially functional dystrophin protein. A practical problem in thedevelopment of a medicament based on exon-skipping technology is theplurality of mutations that may result in a deficiency in functionaldystrophin protein in the cell. Despite the fact that already multipledifferent mutations can be corrected for by the skipping of a singleexon, this plurality of mutations, requires the generation of a seriesof different pharmaceuticals as for different mutations different exonsneed to be skipped. An advantage of an oligonucleotide or of acomposition comprising at least two distinct oligonucleotide as laterdefined herein capable of inducing skipping of two or more exons, isthat more than one exon can be skipped with a single pharmaceutical.This property is not only practically very useful in that only a limitednumber of pharmaceuticals need to be generated for treating manydifferent DMD or particular, severe BMD mutations. Another option nowopen to the person skilled in the art is to select particularlyfunctional restructured dystrophin proteins and produce compoundscapable of generating these preferred dystrophin proteins. Suchpreferred end results are further referred to as mild phenotypedystrophins.

Dose ranges of oligonucleotide according to the invention are preferablydesigned on the basis of rising dose studies in clinical trials (in vivouse) for which rigorous protocol requirements exist. A molecule or anoligonucleotide as defined herein may be used at a dose which is rangedbetween 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg.

In a preferred embodiment, a concentration of an oligonucleotide asdefined herein, which is ranged between 0.1 nM and 1 μM is used.Preferably, this range is for in vitro use in a cellular model such asmuscular cells or muscular tissue. More preferably, the concentrationused is ranged between 0.3 to 400 nM, even more preferably between 1 to200 nM. If several oligonucleotides are used, this concentration or dosemay refer to the total concentration or dose of oligonucleotides or theconcentration or dose of each oligonucleotide added.

The ranges of concentration or dose of oligonucleotide(s) as given aboveare preferred concentrations or doses for in vitro or ex vivo uses. Theskilled person will understand that depending on the oligonucleotide(s)used, the target cell to be treated, the gene target and its expressionlevels, the medium used and the transfection and incubation conditions,the concentration or dose of oligonucleotide(s) used may further varyand may need to be optimised any further.

An oligonucleotide as defined herein for use according to the inventionmay be suitable for administration to a cell, tissue and/or an organ invivo of individuals affected by or at risk of developing DMD or BMD, andmay be administered in vivo, ex vivo or in vitro. Said oligonucleotidemay be directly or indirectly administrated to a cell, tissue and/or anorgan in vivo of an individual affected by or at risk of developing DMDor BMD, and may be administered directly or indirectly in vivo, ex vivoor in vitro. As Duchenne and Becker muscular dystrophy have a pronouncedphenotype in muscle cells, it is preferred that said cells are musclecells, it is further preferred that said tissue is a muscular tissueand/or it is further preferred that said organ comprises or consists ofa muscular tissue. A preferred organ is the heart. Preferably, saidcells comprise a gene encoding a mutant dystrophin protein. Preferably,said cells are cells of an individual suffering from DMD or BMD.

An oligonucleotide of the invention may be indirectly administratedusing suitable means known in the art. An oligonucleotide may forexample be provided to an individual or a cell, tissue or organ of saidindivival in the form of an expression vector wherein the expressionvector encodes a transcript comprising said oligonucleotide. Theexpression vector is preferably introduced into a cell, tissue, organ orindividual via a gene delivery vehicle. In a preferred embodiment, thereis provided a viral-based expression vector comprising an expressioncassette or a transcription cassette that drives expression ortranscription of a molecule as identified herein. A preferred deliveryvehicle is a viral vector such as an adeno-associated virus vector(AAV), or a retroviral vector such as a lentivirus vector^(4, 51, 52)and the like. Also, plasmids, artificial chromosomes, plasmids suitablefor targeted homologous recombination and integration in the humangenome of cells may be suitably applied for delivery of anoligonucleotide as defined herein. Preferred for the current inventionare those vectors wherein transcription is driven from PolIII promoters,and/or wherein transcripts are in the form fusions with U1 or U7transcripts, which yield good results for delivering small transcripts.It is within the skill of the artisan to design suitable transcripts.Preferred are PolIII driven transcripts. Preferably, in the form of afusion transcript with an U1 or U7 transcript^(4, 51, 52). Such fusionsmay be generated as described^(53, 54). The oligonucleotide may bedelivered as is. However, the oligonucleotide may also be encoded by theviral vector. Typically, this is in the form of an RNA transcript thatcomprises the sequence of the oligonucleotide in a part of thetranscript.

Improvements in means for providing an individual or a cell, tissue,organ of said individual with an oligonucleotide and/or an equivalentthereof, are anticipated considering the progress that has already thusfar been achieved. Such future improvements may of course beincorporated to achieve the mentioned effect on restructuring of mRNAusing a method of the invention. An oligonucleotide and/or an equivalentthereof can be delivered as is to an individual, a cell, tissue or organof said individual. When administering an oligonucleotide and/or anequivalent thereof, it is preferred that an oligonucleotide and/or anequivalent thereof is dissolved in a solution that is compatible withthe delivery method. For intravenous, subcutaneous, intramuscular,intrathecal and/or intraventricular administration it is preferred thatthe solution is a physiological salt solution. Particularly preferred inthe invention is the use of an excipient that will aid in delivery ofeach of the constituents as defined herein to a cell and/or into a cell,preferably a muscle cell. Preferred are excipients capable of formingcomplexes, nanoparticles, micelles, vesicles and/or liposomes thatdeliver each constituent as defined herein, complexed or trapped in avesicle or liposome through a cell membrane. Many of these excipientsare known in the art. Suitable excipients comprise polyethylenimine(PEI), or similar cationic polymers, including polypropyleneimine orpolyethylenimine copolymers (PECs) and derivatives,

synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsidproteins that are capable of self assembly into particles that candeliver each constitutent as defined herein to a cell, preferably amuscle cell. Such excipients have been shown to efficiently deliver anoligonucleotide such as antisense nucleic acids to a wide variety ofcultured cells, including muscle cells. Their high transfectionpotential is combined with an excepted low to moderate toxicity in termsof overall cell survival. The ease of structural modification can beused to allow further modifications and the analysis of their further(in vivo) nucleic acid transfer characteristics and toxicity.

Lipofectin represents an example of a liposomal transfection agent. Itconsists of two lipid components, a cationic lipid N-[1-(2,3dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAPwhich is the methylsulfate salt) and a neutral lipiddioleoylphosphatidylethanolamine (DOPE). The neutral component mediatesthe intracellular release. Another group of delivery systems arepolymeric nanoparticles.

Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, whichare well known as DNA transfection reagent can be combined withbutylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulatecationic nanoparticles that can deliver each constituent as definedherein, preferably an oligonucleotide across cell membranes into cells.

In addition to these common nanoparticle materials, the cationic peptideprotamine offers an alternative approach to formulate an oligonucleotidewith colloids. This colloidal nanoparticle system can form so calledproticles, which can be prepared by a simple self-assembly process topackage and mediate intracellular release of an oligonucleotide. Theskilled person may select and adapt any of the above or othercommercially available alternative excipients and delivery systems topackage and deliver an oligonucleotide for use in the current inventionto deliver it for the treatment of Duchenne Muscular Dystrophy or BeckerMuscular Dystrophy in humans.

In addition, an oligonucleotide could be covalently or non-covalentlylinked to a targeting ligand specifically designed to facilitate theuptake in to the cell, cytoplasm and/or its nucleus. Such ligand couldcomprise (i) a compound (including but not limited to peptide(-like)structures) recognising cell, tissue or organ specific elementsfacilitating cellular uptake and/or (ii) a chemical compound able tofacilitate the uptake in to cells and/or the intracellular release of anoligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, an oligonucleotide is formulatedin a composition or a medicament or a composition, which is providedwith at least an excipient and/or a targeting ligand for delivery and/ora delivery device thereof to a cell and/or enhancing its intracellulardelivery. Accordingly, the invention also encompasses a pharmaceuticallyacceptable composition comprising an oligonucleotide and furthercomprising at least one excipient and/or a targeting ligand for deliveryand/or a delivery device of said oligonucleotide to a cell and/orenhancing its intracellular delivery. It is to be understood that if acomposition comprises an additional constituent such as an adjunctcompound as later defined herein, each constituent of the compositionmay not be formulated in one single combination or composition orpreparation. Depending on their identity, the skilled person will knowwhich type of formulation is the most appropriate for each constituentas defined herein. In a preferred embodiment, the invention provides acomposition or a preparation which is in the form of a kit of partscomprising an oligonucleotide and a further adjunct compound as laterdefined herein.

A preferred oligonucleotide is for preventing or treating DuchenneMuscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in anindividual. An individual, which may be treated using an oligonucleotideof the invention may already have been diagnosed as having a DMD or aBMD. Alternatively, an individual which may be treated using anoligonucleotide of the invention may not have yet been diagnosed ashaving a DMD or a BMD but may be an individual having an increased riskof developing a DMD or a BMD in the future given his or her geneticbackground. A preferred individual is a human being.

Composition

In a further aspect, there is provided a composition comprising anoligonucleotide as defined herein. Preferably, said compositioncomprises at least two distinct oligonucleotide as defined herein. Morepreferably, these two distinct oligonucleotides are designed to skipdistinct two or more exons as earlier defined herein for multi-exonskipping.

In a preferred embodiment, said composition being preferably apharmaceutical composition said pharmaceutical composition comprising apharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.Such a pharmaceutical composition may comprise any pharmaceuticallyacceptable carrier, filler, preservative, adjuvant, solubilizer, diluentand/or excipient is also provided. Such pharmaceutically acceptablecarrier, filler, preservative, adjuvant, solubilizer, diluent and/orexcipient may for instance be found in Remington: The Science andPractice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams& Wilkins, 2000. Each feature of said composition has earlier beendefined herein.

If several oligonucleotides are used, concentration or dose alreadydefined herein may refer to the total concentration or dose of alloligonucleotides used or the concentration or dose of eacholigonucleotide used or added. Therefore in one embodiment, there isprovided a composition wherein each or the total amount ofoligonucleotide used is dosed in an amount ranged between 0.5 mg/kg and10 mg/kg.

A preferred composition additionally comprises:

-   -   a) an adjunct compound for reducing inflammation, preferably for        reducing muscle tissue inflammation, and/or    -   b) an adjunct compound for improving muscle fiber function,        integrity and/or survival and/or    -   c) a compound exhibiting readthrough activity.

It has surprisingly been found that the skipping frequency of adystrophin exon from a pre-MRNA comprising said exon, when using anoligonucleotide directed toward the exon or to one or both splice sitesof said exon, is enhanced if cells expressing said pre-mRNA are alsoprovided with an adjunct compound for reducing inflammation, preferablyfor reducing muscle tissue inflammation, and/or an adjunct compound forimproving muscle fiber function, integrity and/or survival. The enhancedskipping frequency also increases the level of functional dystrophinprotein produced in a muscle cell of a DMD or BMD individual.

According to the present invention, even when a dystrophin proteindeficiency has been restored in a DMD patient by administering anoligonucleotide of the invention, the presence of tissue inflammationand damaged muscle cells still continues to contribute to the symptomsof DMD. Hence, even though the cause of DMD—i.e. a dysfunctionaldystrophin protein—is alleviated, treatment of DMD is still furtherimproved by additionally using an adjunct therapy according to thepresent invention. Furthermore, the present invention provides theinsight that a reduction of inflammation does not result in significantreduction of AON uptake by muscle cells. This is surprising because, ingeneral, inflammation enhances the trafficking of cells, blood and othercompounds. As a result, AON uptake/delivery is also enhanced duringinflammation. Hence, before the present invention it would be expectedthat an adjunct therapy counteracting inflammation involves the risk ofnegatively influencing AON therapy. This, however, appears not to be thecase.

An adjunct compound for reducing inflammation comprises any therapywhich is capable of at least in part reducing inflammation, preferablyinflammation caused by damaged muscle cells. Said adjunct compound ismost preferably capable of reducing muscle tissue inflammation.Inflammation is preferably assessed by detecting an increase in thenumber of infiltrating immune cells such as neutrophils and/or mastcells and/or dendritic cells and/or lymphocytes in muscle tissuesuspected to be dystrophic. This assessment is preferably carried out incross-sections of a biopsy⁵⁷ of muscle tissue suspected to be dystrophicafter having specifically stained immune cells as identified above. Thequantification is preferably carried out under the microscope. Reducinginflammation is therefore preferably assessed by detecting a decrease inthe number of immune cells in a cross-section of muscle tissue suspectedto be dystrophic. Detecting a decrease preferably means that the numberof at least one sort of immune cells as identified above is decreased ofat least 1%, 2%, 3%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more compared to the number of a correspondingimmune cell in a same individual before treatment. Most preferably, noinfiltrating immune cells are detected in cross-sections of said biopsy.

An adjunct compound for improving muscle fiber function, integrityand/or survival comprises any therapy, which is capable of measurablyenhancing muscle fiber function, integrity and/or survival as comparedto an otherwise similar situation wherein said adjunct compound is notpresent. The improvement of muscle fiber function, integrity and/orsurvival may be assessed using at least one of the following assays: adetectable decrease of creatine kinase in blood, a detectable decreaseof necrosis of muscle fibers in a biopsy cross-section of a musclesuspected to be dystrophic, and/or a detectable increase of thehomogeneity of the diameter of muscle fibers in a biopsy cross-sectionof a muscle suspected to be dystrophic. Each of these assays is known tothe skilled person.

Creatine kinase may be detected in blood as described in 57. Adetectable decrease in creatine kinase may mean a decrease of 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to theconcentration of creatine kinase in a same individual before treatment.

A detectable decrease of necrosis of muscle fibers is preferablyassessed in a muscle biopsy, more preferably as described in 57 usingbiopsy cross-sections. A detectable decrease of necrosis may be adecrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more ofthe area wherein necrosis has been identified using biopsycross-sections. The decrease is measured by comparison to the necrosisas assessed in a same individual before treatment.

A detectable increase of the homogeneity of the diameter of a musclefiber is preferably assessed in a muscle biopsy cross-section, morepreferably as described in 57.

In one embodiment, an adjunct compound for increasing turnover ofdamaged muscle cells is used. An adjunct compound for increasingturnover of damaged muscle cells comprises any therapy, which is capableof at least in part inducing and/or increasing turnover of damagedmuscle cells. Damaged muscle cells are muscle cells, which havesignificantly less clinically measurable functionality than a healthy,intact muscle cell. In the absence of dystrophin, mechanical stressleads to sarcolemmal ruptures, causing an uncontrolled influx of calciuminto the muscle fiber interior, thereby triggering calcium-activatedproteases and fiber necrosis, resulting in damaged muscle cells.Increasing turnover of damaged muscle cells means that damaged musclecells are more quickly broken down and/or removed as compared to asituation wherein turnover of damaged muscle cells is not increased.Turnover of damaged muscle cells is preferably assessed in a musclebiopsy, more preferably as described in 57 using a cross-section of abiopsy. A detectable increase of turnover may be an increase of 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area whereinturnover has been identified using a biopsy cross-section. The increaseis measured by comparison to the turnover as assessed in a sameindividual before treatment.

Without wishing to be bound to theory, it is believed that increasingturnover of muscle cells is preferred because this reduces inflammatoryresponses.

According to the present invention, a composition of the inventionfurther comprising an adjunct therapy for reducing inflammation,preferably for reducing muscle tissue inflammation in an individual, isparticularly suitable for use as a medicament. Such composition is evenbetter capable of alleviating one or more symptom(s) of DuchenneMuscular Dystrophy or Becker Muscular Dystrophy as compared to acombination not comprising said adjunct compound. This embodiment alsoenhances the skipping frequency of a dystrophin exon from a pre-mRNAcomprising said exon, when using an oligonucleotide directed toward theexon or to one or both splice sites of said exon. The enhanced skippingfrequency also increases the level of functional dystrophin proteinproduced in a muscle cell of a DMD or BMD individual.

Further provided is therefore a composition further comprising anadjunct compound for reducing inflammation, preferably for reducingmuscle tissue inflammation in said individual, for use as a medicament,preferably for treating or preventing counteracting DMD. In oneembodiment, said composition is used in order to alleviate one or moresymptom(s) of a severe form of BMD wherein a very short dystrophinprotein or altered or truncated dystrophin mRNA or protein is formedwhich is not sufficiently functional.

Preferred adjunct compound for reducing inflammation include a steroid,a TNFα inhibitor, a source of mIGF-1 and/or an antioxidant. However, anyother compound able to reduce inflammation as defined herein is alsoencompassed within the present invention. Each of these compounds islater on extensively presented. Each of the compounds extensivelypresented may be used separately or in combination with each otherand/or in combination with one or more of the adjunct compounds used forimproving muscle fiber function, integrity and/or survival.

Furthermore, a composition comprising an adjunct therapy for improvingmuscle fiber function, integrity and/or survival in an individual isparticularly suitable for use as a medicament, preferably for treatingor preventing DMD. Such composition is even better capable ofalleviating one or more symptom(s) of Duchenne Muscular Dystrophy ascompared to a composition not comprising said adjunct compound.

Preferred adjunct compounds for improving muscle fiber function,integrity and/or survival include an ion channel inhibitor, a proteaseinhibitor, L-arginine and/or an angiotensin II type I receptor blocker.However, any other compound able to improving muscle fiber function,integrity and/or survival as defined herein is also encompassed withinthe present invention. Each of these compounds is later on extensivelypresented. Each of the compounds extensively presented may be usedseparately or in combination with each other and/or in combination withone or more of the adjunct compounds used for reducing inflammation.

In a particularly preferred embodiment, a composition further comprisesa steroid. Such composition results in significant alleviation of DMDsymptoms. This embodiment also enhances the skipping frequency of adystrophin exon from a pre-mRNA comprising said exon, when using anoligonucleotide directed toward the exon or to one or both splice sitesof said exon. The enhanced skipping frequency also increases the levelof functional dystrophin protein produced in a muscle cell of a DMD orBMD individual.

In one embodiment, said composition is used in order to alleviate one ormore symptom(s) of a severe form of BMD wherein a very short dystrophinprotein is formed which is not sufficiently functional.

A steroid is a terpenoid lipid characterized by a carbon skeleton withfour fused rings, generally arranged in a 6-6-6-5 fashion. Steroids varyby the functional groups attached to these rings and the oxidation stateof the rings. Steroids include hormones and drugs, which are usuallyused to relieve swelling and inflammation, such as for instanceprednisone, dexamethasone and vitamin D.

According to the present invention, supplemental effects of adjunctsteroid therapy in DMD patients include reduction of tissueinflammation, suppression of cytotoxic cells, and improved calciumhomeostasis. Most positive results are obtained in younger boys.Preferably, the steroid is a corticosteroid, more preferably, aglucocorticosteroid. Preferably, prednisone steroids such as prednisone,prednizolone or deflazacort are used in a combination according to theinvention²¹. Dose ranges of steroid or of a glucocorticosteroid to beused in the therapeutic applications as described herein are designed onthe basis of rising dose studies in clinical trials for which rigorousprotocol requirements exist. The usual doses are 0.5-1.0 mg/kg/day,preferably 0.75 mg/kg/day for prednisone and prednisolone, and 0.4-1.4mg/kg/day, preferably 0.9 mg/kg/day for deflazacort.

In one embodiment, a steroid is administered to said individual prior toadministering a composition as earlier defined herein. In thisembodiment, it is preferred that said steroid is administered at leastone day, more preferred at least one week, more preferred at least twoweeks, more preferred at least three weeks prior to administering saidcomposition.

In another preferred embodiment, a combination further comprises atumour necrosis factor-alpha (TNFα) inhibitor. Tumour necrosisfactor-alpha (TNFα) is a pro-inflammatory cytokine that stimulates theinflammatory response. Pharmacological blockade of TNFα activity withthe neutralising antibody infliximab (Remicade) is highly effectiveclinically at reducing symptoms of inflammatory diseases. In mdx mice,both infliximab and etanercept delay and reduce the necrosis ofdystrophic muscle^(21, 25), with additional physiological benefits onmuscle strength, chloride channel function and reduced CK levels beingdemonstrated in chronically treated exercised adult mdx mice²⁶. Suchhighly specific anti-inflammatory drugs designed for use in otherclinical conditions, are attractive alternatives to the use of steroidsfor DMD. In one embodiment, the use of a TNFα inhibitor is limited toperiods of intensive muscle growth in boys when muscle damage anddeterioration are especially pronounced.

A composition further comprising a TNFα inhibitor for use as amedicament is also provided. In one embodiment, said composition is usedin order to alleviate one or more symptom(s) of a severe form of BMDwherein a very short dystrophin protein is formed which is notsufficiently functional. A preferred TNFα inhibitor is a dimeric fusionprotein consisting of the extracellular ligand-binding domain of thehuman p75 receptor of TNFα linked to the Fc portion of human IgG1. Amore preferred TNFα inhibitor is ethanercept (Amgen, America)²⁶. Theusual doses of ethanercept is about 0.2 mg/kg, preferably about 0.5mg/kg twice a week. The administration is preferably subcutaneous.

In another preferred embodiment, a composition of the invention furthercomprises a source of mIGF-1. As defined herein, a source of IGF-1preferably encompasses mIGF-1 itself, a compound able of enhancingmIGF-1 expression and/or activity. Enhancing is herein synonymous withincreasing. Expression of mIGF-1 is synonymous with amount of mIGF-1.mIGF-1 promotes regeneration of muscles through increase in satellitecell activity, and reduces inflammation and fibrosis²⁷. Local injury ofmuscle results in increased mIGF-1 expression. In transgenic mice withextra IGF-1 genes, muscle hypertrophy and enlarged muscle fibers areobserved²⁷. Similarly, transgenic mdx mice show reduced muscle fiberdegeneration²⁸. Upregulation of the mIGF-1 gene and/or administration ofextra amounts of mIGF-1 protein or a functional equivalent thereof(especially the mIGF-1 Ea isoform [as described in 27, human homologIGF-1 isoform 4: SEQ ID NO: 577]) thus promotes the effect of other,preferably genetic, therapies for DMD, including antisense-induced exonskipping. The additional mIGF-1 levels in the above mentioned transgenicmice do not induce cardiac problems nor promote cancer, and have nopathological side effects. As stated before, the amount of mIGF-1 is forinstance increased by enhancing expression of the mIGF-1 gene and/or byadministration of mIGF-1 protein and/or a functional equivalent thereof(especially the mIGF-1 Ea isoform [as described in 27, human homologIGF-1 isoform 4: SEQ ID NO: 577]). A composition of the inventionfurther preferably comprises mIGF-1, a compound capable of enhancingmIGF-1 expression and/or an mIGF-1 activity, for use as a medicament isalso provided. Said medicament is preferably for alleviating one or moresymptom(s) of DMD. In one embodiment, such composition is used in orderto alleviate one or more symptom(s) of a severe form of BMD wherein avery short dystrophin protein is formed which is not sufficientlyfunctional.

Within the context of the invention, an increased amount or activity ofmIGF-1 may be reached by increasing the gene expression level of anIGF-1 gene, by increasing the amount of a corresponding IGF-1 proteinand/or by increasing an activity of an IGF1-protein. A preferred mIGF-1protein has been earlier defined herein. An increase of an activity ofsaid protein is herein understood to mean any detectable change in abiological activity exerted by said protein or in the steady state levelof said protein as compared to said activity or steady-state in aindividual who has not been treated. Increased amount or activity ofmIGF-1 is preferably assessed by detection of increased expression ofmuscle hypertrophy biomarker GATA-2 (as described in 27).

Gene expression level is preferably assessed using classical molecularbiology techniques such as (real time) PCR, arrays or Northern analysis.A steady state level of a protein is determined directly by quantifyingthe amount of a protein. Quantifying a protein amount may be carried outby any known technique such as Western blotting or immunoassay using anantibody raised against a protein. The skilled person will understandthat alternatively or in combination with the quantification of a geneexpression level and/or a corresponding protein, the quantification of asubstrate of a corresponding protein or of any compound known to beassociated with a function or activity of a corresponding protein or thequantification of said function or activity of a corresponding proteinusing a specific assay may be used to assess the alteration of anactivity or steady state level of a protein.

In the invention, an activity or steady-state level of a said proteinmay be altered at the level of the protein itself, e.g. by providing aprotein to a cell from an exogenous source.

Preferably, an increase or an up-regulation of the expression level of asaid gene means an increase of at least 5% of the expression level ofsaid gene using arrays. More preferably, an increase of the expressionlevel of said gene means an increase of at least 10%, even morepreferably at least 20%, at least 30%, at least 40%, at least 50%, atleast 70%, at least 90%, at least 150% or more. In another preferredembodiment, an increase of the expression level of said protein means anincrease of at least 5% of the expression level of said protein usingWestern blotting and/or using ELISA or a suitable assay. Morepreferably, an increase of the expression level of a protein means anincrease of at least 10%, even more preferably at least 20%, at least30%, at least 40%, at least 50%, at least 70%, at least 90%, at least150% or more.

In another preferred embodiment, an increase of a polypeptide activitymeans an increase of at least 5% of a polypeptide activity using asuitable assay. More preferably, an increase of a polypeptide activitymeans an increase of at least 10%, even more preferably at least 20%, atleast 30%, at least 40%, at least 50%, at least 70%, at least 90%, atleast 150% or more. The increase is preferably assessed by comparison tocorresponding activity in the individual before treatment.

A preferred way of providing a source of mIGF1 is to introduce atransgene encoding mIGF1, preferably an mIGF-1 Ea isoform (as describedin 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577), more preferablyin an AAV vector as later defined herein. Such source of mIGF1 isspecifically expressed in muscle tissue as described in mice in 27.

In another preferred embodiment, a composition further comprises anantioxidant. Oxidative stress is an important factor in the progressionof DMD and promotes chronic inflammation and fibrosis²⁹. The mostprevalent products of oxidative stress, the peroxidized lipids, areincreased by an average of 35% in Duchenne boys. Increased levels of theenzymes superoxide dismutase and catalase reduce the excessive amount offree radicals causing these effects. In fact, a dietary supplementProtandim® (LifeVantage) was clinically tested and found to increaselevels of superoxide dismutase (up to 30%) and catalase (up to 54%),which indeed significantly inhibited the peroxidation of lipids in 29healthy persons³⁰. Such effective management of oxidative stress thuspreserves muscle quality and so promotes the positive effect of DMDtherapy. Idebenone is another potent antioxidant with a chemicalstructure derived from natural coenzyme Q10. It protects mitochondriawhere adenosine triphosphate, ATP, is generated by oxidativephosphorylation. The absence of dystrophin in DMD negatively affectsthis process in the heart, and probably also in skeletal muscle.Idebenone was recently applied in clinical trials in the US and Europedemonstrating efficacy on neurological aspects of Friedreich's Ataxia³¹.A phase-IIa double-blind, placebo-controlled randomized clinical trialwith Idebenone has recently been started in Belgium, including 21Duchenne boys at 8 to 16 years of age. The primary objective of thisstudy is to determine the effect of Idebenone on heart muscle function.In addition, several different tests will be performed to detect thepossible functional benefit on muscle strength in the patients. Wheneffective, Idebenone is a preferred adjunct compound for use in acombination according to the present invention in order to enhance thetherapeutic effect of DMD therapy, especially in the heart. Acomposition further comprising an antioxidant for use as a medicament isalso provided. Said medicament is preferably for alleviating one or moresymptom(s) of DMD. In one embodiment, said composition is used in orderto alleviate one or more symptom(s) of a severe form of BMD wherein avery short dystrophin protein is formed which is not sufficientlyfunctional. Depending on the identity of the antioxidant, the skilledperson will know which quantities are preferably used. An antioxidantmay include bacoside, silymarin, curcumin and/or a polyphenol.Preferably, a polyphenol is or comprises epigallocatechin-3-gallate(EGCG). Preferably, an antioxidant is a mixture of antioxidants as thedietary supplement Protandim® (LifeVantage). A daily capsule of 675 mgof Protandim® comprises 150 mg of B. monniera (45% bacosides), 225 mg ofS. marianum (70-80% silymarin), 150 mg of W. somnifera powder, 75 mggreen tea (98% polyphenols wherein 45% EGCG) and 75 mg turmeric (95%curcumin).

In another preferred embodiment, a composition further comprises an ionchannel inhibitor. The presence of damaged muscle membranes in DMDdisturbs the passage of calcium ions into the myofibers, and theconsequently disrupted calcium homeostasis activates many enzymes, e.g.proteases, that cause additional damage and muscle necrosis. Ionchannels that directly contribute to the pathological accumulation ofcalcium in dystrophic muscle are potential targets for adjunct compoundsto treat DMD. There is evidence that some drugs, such as pentoxifylline,block exercise-sensitive calcium channels³² and antibiotics that blockstretch activated channels reduce myofibre necrosis in mdx mice and CKlevels in DMD boys³³. A composition further comprising an ion channelinhibitor for use as a medicament is also provided. Said medicament ispreferably for alleviating one or more symptom(s) of DMD. In oneembodiment, said composition is used in order to alleviate one or moresymptom(s) of a severe form of BMD wherein a very short dystrophinprotein is formed which is not sufficiently functional.

Preferably, an ion channel inhibitor of the class of xanthines is used.More preferably, said xanthines are derivatives of methylxanthines, andmost preferably, said methylxanthine derivates are chosen from the groupconsisting of pentoxifylline, furafylline, lisofylline, propentofylline,pentifylline, theophylline, torbafylline, albifylline, enprofylline andderivatives thereof. Most preferred is the use of pentoxifylline. Ionchannel inhibitors of the class of xanthines enhance the skippingfrequency of a dystrophin exon from a pre-mRNA comprising said exon,when using an oligonucleotide directed toward the exon or to one or bothsplice sites of said exon. The enhanced skipping frequency alsoincreases the level of functional dystrophin protein produced in amuscle cell of a DMD or BMD individual.

Depending on the identity of the ion channel inhibitor, the skilledperson will know which quantities are preferably used. Suitable dosagesof pentoxifylline are between 1 mg/kg/day to 100 mg/kg/day, preferreddosages are between 10 mg/kg/day to 50 mg/kg/day. Typical dosages usedin humans are 20 mg/kg/day.

In one embodiment, an ion channel inhibitor is administered to saidindividual prior to administering a composition comprising anoligonucleotide. In this embodiment, it is preferred that said ionchannel inhibitor is administered at least one day, more preferred atleast one week, more preferred at least two weeks, more preferred atleast three weeks prior to administering a composition comprising anoligonucleotide.

In another preferred embodiment, a composition further comprises aprotease inhibitor. Calpains are calcium-activated proteases that areincreased in dystrophic muscle and account for myofiber degeneration.Calpain inhibitors such as calpastatin, leupeptin³⁴, calpeptin, calpaininhibitor III, or PD150606 are therefore applied to reduce thedegeneration process. A new compound, BN 82270 (Ipsen) that has dualaction as both a calpain inhibitor and an antioxidant increased musclestrength, decreased serum CK and reduced fibrosis of the mdx diaphragm,indicating a therapeutic effect with this new compound³⁵. Anothercompound of Leupeptin/Carnitine (Myodur) has recently been proposed forclinical trials in DMD patients.

MG132 is another proteasomal inhibitor that has shown to reduce musclemembrane damage, and to ameliorate the histopathological signs ofmuscular dystrophy³⁶. MG-132 (CBZ-leucyl-leucyl-leucinal) is acell-permeable, proteasomal inhibitor (Ki=4 nM), which inhibits NFkappaBactivation by preventing IkappaB degradation (IC50=3 μM). In addition,it is a peptide aldehyde that inhibits ubiquitin-mediated proteolysis bybinding to and inactivating 20S and 26S proteasomes. MG-132 has shown toinhibit the proteasomal degradation of dystrophin-associated proteins inthe dystrophic mdx mouse model³⁶. This compound is thus also suitablefor use as an adjunct pharmacological compound for DMD. A compositionfurther comprising a protease inhibitor for use as a medicament is alsoprovided. Said medicament is preferably for alleviating one or moresymptom(s) of DMD. In one embodiment, said combination is used in orderto alleviate one or more symptom(s) of a severe form of BMD wherein avery short dystrophin protein is formed which is not sufficientlyfunctional. Depending on the identity of the protease inhibitor, theskilled person will know which quantities are preferably used.

In another preferred embodiment, a composition further comprisesL-arginine. Dystrophin-deficiency is associated with the loss of theDGC-complex at the fiber membranes, including neuronal nitric oxidesynthase (nNOS). Expression of a nNOS transgene in mdx mice greatlyreduced muscle membrane damage. Similarly, administration of L-arginine(the substrate for nitric oxide synthase) increased NO production andupregulated utrophin expression in mdx mice. Six weeks of L-argininetreatment improved muscle pathology and decreased serum CK in mdxmice³⁷. The use of L-arginine as a further constituent in a compositionof the invention has not been disclosed.

A composition further comprising L-arginine for use as a medicament isalso provided. Said medicament is preferably for alleviating one or moresymptom(s) of DMD. In one embodiment, said composition is used in orderto alleviate one or more symptom(s) of a severe form of BMD wherein avery short dystrophin protein is formed which is not sufficientlyfunctional.

In another preferred embodiment, a composition further comprisesangiotensin II type 1 receptor blocker Losartan, which normalizes musclearchitecture, repair and function, as shown in the dystrophin-deficientmdx mouse model²³. A composition further comprising angiotensin II type1 receptor blocker Losartan for use as a medicament is also provided.Said medicament is preferably for alleviating one or more symptom(s) ofDMD. In one embodiment, said composition is used in order to alleviateone or more symptom(s) of a severe form of BMD wherein a very shortdystrophin protein is formed which is not sufficiently functional.Depending on the identity of the angiotensin II type 1 receptor blocker,the skilled person will know which quantities are preferably used.

In another preferred embodiment, a composition further comprises anangiotensin-converting enzyme (ACE) inhibitor, preferably perindopril.ACE inhibitors are capable of lowering blood pressure. Early initiationof treatment with perindopril is associated with a lower mortality inDMD patients²². A composition further comprising an ACE inhibitor,preferably perindopril for use as a medicament is also provided. Saidmedicament is preferably for alleviating one or more symptom(s) of DMD.In one embodiment, said composition is used in order to alleviate one ormore symptom(s) of a severe form of BMD wherein a very short dystrophinprotein is formed which is not sufficiently functional. The usual dosesof an ACE inhibitor, preferably perindopril are about 2 to 4 mg/day²².In a more preferred embodiment, an ACE inhibitor is combined with atleast one of the previously identified adjunct compounds.

In another preferred embodiment, a composition further comprises acompound exhibiting a readthrough activity. A compound exhibiting areadthrough activity may be any compound, which is able to suppress astop codon. For 20% of DMD patients, the mutation in the dystrophin geneis comprising a point mutation, of which 13% is a nonsense mutation. Acompound exhibiting a readthrough activity or which is able to suppressa stop codon is a compound which is able to provide an increased amountof a functional dystrophin mRNA or protein and/or a decreased amount ofan aberrant or truncated dystrophin mRNA or protein. Increasedpreferably means increased of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% or more. Decreased preferably means decreased of at least 1%, 2%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100% or more. An increase or a decrease of saidprotein is preferably assessed in a muscular tissue or in a muscularcell of an individual by comparison to the amount present in saidindividual before treatment with said compound exhibiting a readthroughactivity. Alternatively, the comparison can be made with a musculartissue or cell of said individual, which has not yet been treated withsaid compound in case the treatment is local. The assessment of anamount at the protein level is preferably carried out using western blotanalysis.

Preferred compounds exhibiting a readthrough activity comprise orconsist of aminoglycosides, including, but not limited to, geneticin(G418), paromomycin, gentamycin and/or3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid), andderivatives thereof (references 64, 65). A more preferred compoundexhibiting a readthrough activity comprises or consists of PTC124™,and/or a functional equivalent thereof. PTC124™ is a registeredtrademark of PTC Therapeutics, Inc. South Plainfield, N.J.3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known asPTC124™ (references 16, 17) belongs to a new class of small moleculesthat mimics at lower concentrations the readthrough activity ofgentamicin (reference 55). A functional equivalent of3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or ofgentamicin is a compound which is able to exhibit a readthrough activityas earlier defined herein. Most preferably, a compound exhibiting areadthrough activity comprises or consists of gentamycin and/or3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known asPTC124™. A composition further comprising a compound exhibiting areadthrough activity, preferably comprising or consisting of gentamycinand/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) for useas a medicament is also provided. Said medicament is preferably foralleviating one or more symptom(s) of DMD. In one embodiment, saidcomposition is used in order to alleviate one or more symptom(s) of asevere form of BMD wherein a very short dystrophin protein is formedwhich is not sufficiently functional. The usual doses of a compoundexhibiting a readthrough activity, preferably3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or ofgentamicin are ranged between 3 mg/kg/day to 200 mg/kg/day, preferreddosages are between 10 mg/kg to 50 mg/kg per day or twice a day.

In a more preferred embodiment, a compound exhibiting a readthroughactivity is combined with at least one of the previously identifiedadjunct compounds.

In another preferred embodiment, a composition further comprises acompound, which is capable of enhancing exon skipping and/or inhibitingspliceosome assembly and/or splicing. Small chemical compounds, such asfor instance specific indole derivatives, have been shown to selectivelyinhibit spliceosome assembly and splicing³⁸, for instance by interferingwith the binding of serine- and arginine-rich (SR) proteins to theircognate splicing enhancers (ISEs or ESEs) and/or by interfering with thebinding of splicing repressors to silencer sequences (ESSs or ISSs).These compounds are therefore suitable for applying as adjunct compoundsthat enhance exon skipping. A composition further comprising a compoundfor enhancing exon skipping and/or inhibiting spliceosome assemblyand/or splicing for use as a medicament is also provided. Saidmedicament is preferably for alleviating one or more symptom(s) of DMD.In one embodiment, said composition is used in order to alleviate one ormore symptom(s) of a severe form of BMD wherein a very short dystrophinprotein is formed which is not sufficiently functional. Depending on theidentity of the compound, which is capable of enhancing exon skippingand/or inhibiting spliceosome assembly and/or splicing, the skilledperson will know which quantities are preferably used. In a morepreferred embodiment, a compound for enhancing exon skipping and/orinhibiting spliceosome assembly and/or splicing is combined with a ACEinhibitor and/or with any adjunct compounds as identified earlierherein.

The invention thus provides a composition further comprising an adjunctcompound, wherein said adjunct compound comprises a steroid, an ACEinhibitor (preferably perindopril), angiotensin II type 1 receptorblocker Losartan, a tumour necrosis factor-alpha (TNFα) inhibitor, asource of mIGF-1, preferably mIGF-1, a compound for enhancing mIGF-1expression, a compound for enhancing mIGF-1 activity, an antioxidant, anion channel inhibitor, a protease inhibitor, L-arginine, a compoundexhibiting a readthrough activity and/or inhibiting spliceosome assemblyand/or splicing.

In one embodiment an individual is further provided with a functionaldystrophin protein using a vector, preferably a viral vector, comprisinga micro-mini-dystrophin gene. Most preferably, a recombinantadeno-associated viral (rAAV) vector is used. AAV is a single-strandedDNA parvovirus that is non-pathogenic and shows a helper-dependent lifecycle. In contrast to other viruses (adenovirus, retrovirus, and herpessimplex virus), rAAV vectors have demonstrated to be very efficient intransducing mature skeletal muscle. Application of rAAV in classical DMD“gene addition” studies has been hindered by its restricted packaginglimits (<5 kb). Therefore, rAAV is preferably applied for the efficientdelivery of a much smaller micro- or mini-dystrophin gene.Administration of such micro- or mini-dystrophin gene results in thepresence of an at least partially functional dystrophin protein.Reference is made to¹⁸⁻²⁰.

Each constituent of a composition can be administered to an individualin any order. In one embodiment, each constituent is administeredsimultaneously (meaning that each constituent is administered within 10hours, preferably within one hour). This is however not necessary. Inone embodiment at least one adjunct compound is administered to anindividual in need thereof before administration of an oligonucleotide.Alternatively, an oligonucleotide is administered to an individual inneed thereof before administration of at least one adjunct compound.

Use

In a further aspect, there is provided the use of a oligonucleotide orof a composition as defined herein for the manufacture of a medicamentfor preventing or treating Duchenne Muscular Dystrophy or BeckerMuscular Dystrophy in an individual. Each feature of said use hasearlier been defined herein.

A treatment in a use or in a method according to the invention is atleast one week, at least one month, at least several months, at leastone year, at least 2, 3, 4, 5, 6 years or more. Each molecule oroligonucleotide or equivalent thereof as defined herein for useaccording to the invention may be suitable for direct administration toa cell, tissue and/or an organ in vivo of individuals affected by or atrisk of developing DMD or BMD, and may be administered directly in vivo,ex vivo or in vitro. The frequency of administration of anoligonucleotide, composition, compound or adjunct compound of theinvention may depend on several parameters such as the age of thepatient, the mutation of the patient, the number of molecules (i.e.dose), the formulation of said molecule. The frequency may be rangedbetween at least once in two weeks, or three weeks or four weeks or fiveweeks or a longer time period.

Method

In a further aspect, there is provided a method for alleviating one ormore symptom(s) of Duchenne Muscular Dystrophy or Becker MuscularDystrophy in an individual or alleviate one or more characteristic(s) ofa myogenic or muscle cell of said individual, the method comprisingadministering to said individual an oligonucleotide or a composition asdefined herein.

There is further provided a method for enhancing, inducing or promotingskipping of an exon from a dystrophin pre-mRNA in a cell expressing saidpre-mRNA in an individual suffering from Duchenne Muscular Dystrophy orBecker Muscular Dystrophy, the method comprising administering to saidindividual an oligonucleotide or a composition as defined herein.Further provided is a method for increasing the production of afunctional dystrophin protein and/or decreasing the production of anaberrant dystrophin protein in a cell, said cell comprising a pre-mRNAof a dystrophin gene encoding an aberrant dystrophin protein, the methodcomprising providing said cell with an oligonucleotide or composition ofthe invention and allowing translation of mRNA produced from splicing ofsaid pre-mRNA. In one embodiment, said method is performed in vitro, forinstance using a cell culture. Preferably, said method is in vivo.

In this context, increasing the production of a functional dystrophinprotein has been earlier defined herein.

Unless otherwise indicated each embodiment as described herein may becombined with another embodiment as described herein.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a compound or adjunct compoundas defined herein may comprise additional component(s) than the onesspecifically identified, said additional component(s) not altering theunique characteristic of the invention.

In addition, reference to an element by the indefinite article “a” or“an” does not exclude the possibility that more than one of the elementis present, unless the context clearly requires that there be one andonly one of the elements. The indefinite article “a” or “an” thususually means “at least one”.

The word “approximately” or “about” when used in association with anumerical value (approximately 10, about 10) preferably means that thevalue may be the given value of 10 more or less 1% of the value.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety. Each embodimentas identified herein may be combined together unless otherwiseindicated.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . In human control myotubes, PS220 and PS305 both targeting anidentical sequence within exon 45, were directly compared for relativeskipping efficiencies. PS220 reproducibly induced highest levels of exon45 skipping (up to 73%), whereas with PS305 maximum exon 45 skippinglevels of up to 46% were obtained. No exon 45 skipping was observed innon-treated cells. (M: DNA size marker; NT: non-treated cells)

FIG. 2 . Graph showing relative exon 45 skipping levels ofinosine-containing AONs as assessed by RT-PCR analysis. In human controlmyotubes, a series of new AONs, all targeting exon 45 and containing oneinosine for guanosine substitution were tested for relative exon 45skipping efficiencies when compared with PS220 and PS305 (see FIG. 1 ).All new inosine-containing AONs were effective, albeit at variablelevels (between 4% and 25%). PS220 induced highest levels of exon 45skipping (up to 72%), whereas with PS305 maximum exon 45 skipping levelsof up to 63% were obtained. No exon 45 skipping was observed innon-treated cells. (M: DNA size marker; NT: non-treated cells).

EXAMPLES Example 1

Materials and Methods

AON design was based on (partly) overlapping open secondary structuresof the target exon RNA as predicted by the m-fold program, on (partly)overlapping putative SR-protein binding sites as predicted by theESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V.(Leiden, Netherlands), and contain 2′-O-methyl RNA and full-lengthphosphorothioate (PS) backbones.

Tissue Culturing, Transfection and RT-PCR Analysis

Myotube cultures derived from a healthy individual (“human control”)(examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patientcarrying an exon 45 deletion (example 2; exon 46 skipping) wereprocessed as described previously (Aartsma-Rus et al., Neuromuscul.Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For thescreening of AONs, myotube cultures were transfected with 200 nM foreach AON (PS220 and PS305). Transfection reagent UNIFectylin (ProsensaTherapeutics BV, Netherlands) was used, with 2 μl UNIFectylin per μgAON. Exon skipping efficiencies were determined by nested RT-PCRanalysis using primers in the exons flanking the targeted exon 45. PCRfragments were isolated from agarose gels for sequence verification. Forquantification, the PCR products were analyzed using the DNA 1000LabChip Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

Results

DMD Exon 45 Skipping.

Two AONs, PS220 (SEQ ID NO: 76; 5′-UUUGCCGCUGCCCAAUGCCAUCCUG-3′) andPS305 (SEQ ID NO: 557; 5′-UUUGCCICUGCCCAAUGCCAUCCUG-3′) both targetingan identical sequence within exon 45, were directly compared forrelative skipping efficiencies in healthy control myotube cultures.Subsequent RT-PCR and sequence analysis of isolated RNA demonstratedthat both AONs were indeed capable of inducing exon 45 skipping. PS220,consisting a GCCGC stretch, reproducibly induced highest levels of exon45 skipping (up to 73%), as shown in FIG. 1 . However, PS305, which isidentical to PS220 but containing an inosine for a G substitution atposition 4 within that stretch is also effective and leading to exon 45skipping levels of up to 46%. No exon 45 skipping was observed innon-treated cells (NT).

Example 2

Materials and Methods

AON design was based on (partly) overlapping open secondary structuresof the target exon 45 RNA as predicted by the m-fold program, on(partly) overlapping putative SR-protein binding sites as predicted bythe ESE-finder software. AONs were synthesized by Prosensa TherapeuticsB.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA, full-lengthphosphorothioate (PS) backbones and one inosine for guanosinesubstitution.

Tissue Culturing, Transfection and RT-PCR Analysis

Myotube cultures derived from a healthy individual (“human control”)were processed as described previously (Aartsma-Rus et al., Neuromuscul.Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For thescreening of AONs, myotube cultures were transfected with 200 nM foreach AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV,Netherlands) was used, with 2 μl UNIFectylin per μg AON. Exon skippingefficiencies were determined by nested RT-PCR analysis using primers inthe exons flanking the targeted exon 45. PCR fragments were isolatedfrom agarose gels for sequence verification. For quantification, the PCRproducts were analyzed using the DNA 1000 Lab Chip Kit on the Agilent2100 bioanalyzer (Agilent Technologies, USA).

Results

DMD Exon 45 Skipping.

An additional series of AONs targeting exon 45 and containing oneinosine-substitution were tested in healthy control myotube cultures forexon 45 skipping efficiencies, and directly compared to PS220 (withoutinosine; SEQ ID NO: 76)) and PS305 (identical sequence as PS220 but withinosine substitution; SEQ ID NO: 557). Subsequent RT-PCR and sequenceanalysis of isolated RNA demonstrated that all new AONs (PS309 to PS316)were capable of inducing exon 45 skipping between 4% (PS311) and 25%(PS310) as shown in FIG. 2 . When compared to PS220 and PS305, PS220induced highest levels of exon 45 skipping (up to 72%). Of the newinosine-containing AONs PS305 was most effective, showing exon 45skipping levels of up to 63%. No exon 45 skipping was observed innon-treated cells (NT).

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Sequence listing DMD gene amino acid sequence SEQ ID NO 1:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTCQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFITSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTVTTREQILVKHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNFSDLKEKVNAIEREKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIEFCQLLSERLNWLEYQNNIIAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDEVNRLSGLQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKELQTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNKLRKIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKGGLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESVNSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSIQSAQETEKSLHLIQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQRLQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWGKATQKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRKLVE PQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNALKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGICQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVQETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEARTIITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM DMD Gene Exon 51 SEQ ID GUACCUCCAACAUCAAGGAAGAUGG SEQ IDGAGAUGGCAGUUUCCUUAGUAACCA NO 2 NO 39 SEQ ID UACCUCCAACAUCAAGGAAGAUGGCSEQ ID AGAUGGCAGUUUCCUUAGUAACCAC NO 3 NO 40 SEQ IDACCUCCAACAUCAAGGAAGAUGGCA SEQ ID GAUGGCAGUUUCCUUAGUAACCACA NO 4 NO 41SEQ ID CCUCCAACAUCAAGGAAGAUGGCAU SEQ ID AUGGCAGUUUCCUUAGUAACCACAG NO 5NO 42 SEQ ID CUCCAACAUCAAGGAAGAUGGCAUU SEQ ID UGGCAGUUUCCUUAGUAACCACAGGNO 6 NO 43 SEQ ID UCCAACAUCAAGGAAGAUGGCAUUU SEQ IDGGCAGUUUCCUUAGUAACCACAGGU NO 7 NO 44 SEQ ID CCAACAUCAAGGAAGAUGGCAUUUCSEQ ID GCAGUUUCCUUAGUAACCACAGGUU NO 8 NO 45 SEQ IDCAACAUCAAGGAAGAUGGCAUUUCU SEQ ID CAGUUUCCUUAGUAACCACAGGUUG NO 9 NO 46SEQ ID AACAUCAAGGAAGAUGGCAUUUCUA SEQ ID AGUUUCCUUAGUAACCACAGGUUGU NO 10NO 47 SEQ ID ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID GUUUCCUUAGUAACCACAGGUUGUGNO 11 NO 48 SEQ ID CAUCAAGGAAGAUGGCAUUUCUAGU SEQ IDUUUCCUUAGUAACCACAGGUUGUGU NO 12 NO 49 SEQ ID AUCAAGGAAGAUGGCAUUUCUAGUUSEQ ID UUCCUUAGUAACCACAGGUUGUGUC NO 13 NO 50 SEQ IDUCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID UCCUUAGUAACCACAGGUUGUGUCA NO 14 NO 51SEQ ID CAAGGAAGAUGGCAUUUCUAGUUUG SEQ ID CCUUAGUAACCACAGGUUGUGUCAC NO 15NO 52 SEQ ID AAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID CUUAGUAACCACAGGUUGUGUCACCNO 16 NO 53 SEQ ID AGGAAGAUGGCAUUUCUAGUUUGGA SEQ IDUUAGUAACCACAGGUUGUGUCACCA NO 17 NO 54 SEQ ID GGAAGAUGGCAUUUCUAGUUUGGAGSEQTD UAGUAACCACAGGUUGUGUCACCAG NO 18 NO 55 SEQ IDGAAGAUGGCAUUUCUAGUUUGGAGA SEQ ID AGUAACCACAGGUUGUGUCACCAGA NO 19 NO 56SEQ ID AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID GUAACCACAGGUUGUGUCACCAGAG NO 20NO 57 SEQ ID AGAUGGCAUUUCUAGUUUGGAGAUG SEQ ID UAACCACAGGUUGUGUCACCAGAGUNO 21 NO 58 SEQ ID GAUGGCAUUUCUAGUUUGGAGAUGG SEQ IDAACCACAGGUUGUGUCACCAGAGUA NO 22 NO 59 SEQ ID AUGGCAUUUCUAGUUUGGAGAUGGCSEQ ID ACCACAGGUUGUGUCACCAGAGUAA NO 23 NO 60 SEQ IDUGGCAUUUCUAGUUUGGAGAUGGCA SEQ ID CCACAGGUUGUGUCACCAGAGUAAC NO 24 NO 61SEQ ID GGCAUUUCUAGUUUGGAGAUGGCAG SEQ ID CACAGGUUGUGUCACCAGAGUAACA NO 25NO 62 SEQ ID GCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID ACAGGUUGUGUCACCAGAGUAACAGNO 26 NO 63 SEQ ID CAUUUCUAGUUUGGAGAUGGCAGUU SEQ IDCAGGUUGUGUCACCAGAGUAACAGU NO 27 NO 64 SEQ ID AUUUCUAGUUUGGAGAUGGCAGUUUSEQ ID AGGUUGUGUCACCAGAGUAACAGUC NO 28 NO 65 SEQ IDUUUCUAGUUUGGAGAUGGCAGUUUC SEQ ID GGUUGUGUCACCAGAGUAACAGUCU NO 29 NO 66SEQ ID UUCUAGUUUGGAGAUGGCAGUUUCC SEQ ID GUUGUGUCACCAGAGUAACAGUCUG NO 30NO 67 SEQ ID UCUAGUUUGGAGAUGGCAGUUUCCU SEQ ID UUGUGUCACCAGAGUAACAGUCUGANO 31 NO 68 SEQ ID CUAGUUUGGAGAUGGCAGUUUCCUU SEQ IDUGUGUCACCAGAGUAACAGUCUGAG NO 32 NO 69 SEQ ID UAGUUUGGAGAUGGCAGUUUCCUUASEQ ID GUGUCACCAGAGUAACAGUCUGAGU NO 33 NO 70 SEQ IDAGUUUGGAGAUGGCAGUUUCCUUAG SEQ ID UGUCACCAGAGUAACAGUCUGAGUA NO 34 NO 71SEQ ID GUUUGGAGAUGGCAGUUUCCUUAGU SEQ ID GUCACCAGAGUAACAGUCUGAGUAG NO 35NO 72 SEQ ID UUUGGAGAUGGCAGUUUCCUUAGUA SEQ ID UCACCAGAGUAACAGUCUGAGUAGGNO 36 NO 73 SEQ ID UUGGAGAUGGCAGUUUCCUUAGUAA SEQ IDCACCAGAGUAACAGUCUGAGUAGGA NO 37 NO 74 SEQ ID UGGAGAUGGCAGUUUCCUUAGUAACSEQ ID ACCAGAGUAACAGUCUGAGUAGGAG NO 38 NO 75 SEQ ID UCAAGGAAGAUGGCAUUUCUSEQ ID UCAAGGAAGAUGGCAUIUCU NO 539 NO 548 SEQ ID UCAAIGAAGAUGGCAUUUCUSEQ ID UCAAGGAAGAUGGCAUUICU NO 540 NO 549 SEQ ID UCAAGIAAGAUGGCAUUUCUSEQ ID UCAAGGAAGAUGGCAUUUCI NO 541 NO 550 SEQ ID UCAAGGAAIAUGGCAUUUCUSEQ ID UCIAGGAAGAUGGCAUUUCU NO 542 NO 551 SEQ ID UCAAGGAAGAUIGCAUUUCUSEQ ID UCAIGGAAGAUGGCAUUUCU NO 543 NO 552 SEQ ID UCAAGGAAGAUGICAUUUCUSEQ ID UCAAGGIAGAUGGCAUUUCU NO 544 NO 553 SEQ ID ICAAGGAAGAUGGCAUUUCUSEQ ID UCAAGGAIGAUGGCAUUUCU NO 545 NO 554 SEQ ID UCAAGGAAGAIGGCAUUUCUSEQ ID UCAAGGAAGIUGGCAUUUCU NO 546 NO 555 SEQ ID UCAAGGAAGAUGGCAIUUCUSEQ ID UCAAGGAAGAUGGCIUUUCU NO 547 NO 556 DMD Gene Exon 45 SEQ ID UUUGCCG CUGCCCAAUGCCAUCCUG SEQ ID GUUGCAUUCAAUGUUCUGACAACAG NO 76 NO 109 PS220SEQ ID AUUCAAUGUUCUGACAACAGUUUGC SEQ ID UUGCAUUCAAUGUUCUGACAACAGU NO 77NO 110 SEQ ID CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID UGCAUUCAAUGUUCUGACAACAGUUNO 78 NO 111 SEQ ID CAGUUGCAUUCAAUGUUCUGAC SEQ IDGCAUUCAAUGUUCUGACAACAGUUU NO 79 NO 112 SEQ ID AGUUGCAUUCAAUGUUCUGASEQ ID CAUUCAAUGUUCUGACAACAGUUUG NO 80 NO 113 SEQ IDGAUUGCUGAAUUAUUUCUUCC SEQ ID AUUCAAUGUUCUGACAACAGUUUGC NO 81 NO 114SEQ ID GAUUGCUGAAUUAUUUCUUCCCCAG SEQ ID UCAAUGUUCUGACAACAGUUUGCCG NO 82NO 115 SEQ ID AUUGCUGAAUUAUUUCUUCCCCAGU SEQ ID CAAUGUUCUGACAACAGUUUGCCGCNO 83 NO 116 SEQ ID UUGCUGAAUUAUUUCUUCCCCAGUU SEQ IDAAUGUUCUGACAACAGUUUGCCGCU NO 84 NO 117 SEQ ID UGCUGAAUUAUUUCUUCCCCAGUUGSEQ ID AUGUUCUGACAACAGUUUGCCGCUG NO 85 NO 118 SEQ IDGCUGAAUUAUUUCUUCCCCAGUUGC SEQ ID UGUUCUGACAACAGUUUGCCGCUGC NO 86 NO 119SEQ ID CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID GUUCUGACAACAGUUUGCCGCUGCC NO 87NO 120 SEQ ID UGAAUUAUUUCUUCCCCAGUUGCAU SEQ ID UUCUGACAACAGUUUGCCGCUGCCCNO 88 NO 121 SEQ ID GAAUUAUUUCUUCCCCAGUUGCAUU SEQ IDUCUGACAACAGUUUGCCGCUGCCCA NO 89 NO 122 SEQ ID AAUUAUUUCUUCCCCAGUUGCAUUCSEQ ID CUGACAACAGUUUGCCGCUGCCCAA NO 90 NO 123 SEQ IDAUUAUUUCUUCCCCAGUUGCAUUCA SEQ ID UGACAACAGUUUGCCGCUGCCCAAU NO 91 NO 124SEQ ID UUAUUUCUUCCCCAGUUGCAUUCAA SEQ ID GACAACAGUUUGCCGCUGCCCAAUG NO 92NO 125 SEQ ID UAUUUCUUCCCCAGUUGCAUUCAAU SEQ ID ACAACAGUUUGCCGCUGCCCAAUGCNO 93 NO 126 SEQ ID AUUUCUUCCCCAGUUGCAUUCAAUG SEQ IDCAACAGUUUGCCGCUGCCCAAUGCC NO 94 NO 127 SEQ ID UUUCUUCCCCAGUUGCAUUCAAUGUSEQ ID AACAGUUUGCCGCUGCCCAAUGCCA NO 95 NO 128 SEQ IDUUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID ACAGUUUGCCGCUGCCCAAUGCCAU NO 96 NO 129SEQ ID UCUUCCCCAGUUGCAUUCAAUGUUC SEQ ID CAGUUUGCCGCUGCCCAAUGCCAUC NO 97NO 130 SEQ ID CUUCCCCAGUUGCAUUCAAUGUUCU SEQ ID AGUUUGCCGCUGCCCAAUGCCAUCCNO 98 NO 131 SEQ ID UUCCCCAGUUGCAUUCAAUGUUCUG SEQ IDGUUUGCCGCUGCCCAAUGCCAUCCU NO 99 NO 132 SEQ ID UCCCCAGUUGCAUUCAAUGUUCUGASEQ ID UUUGCCGCUGCCCAAUGCCAUCCUG NO 100 NO 133 SEQ IDCCCCAGUUGCAUUCAAUGUUCUGAC SEQ ID UUGCCGCUGCCCAAUGCCAUCCUGG NO 101 NO 134SEQ ID CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID UGCCGCUGCCCAAUGCCAUCCUGGA NO 102NO 135 SEQ ID CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID GCCGCUGCCCAAUGCCAUCCUGGAGNO 103 NO 136 SEQ ID CAGUUGCAUUCAAUGUUCUGACAAC SEQ IDCCGCUGCCCAAUGCCAUCCUGGAGU NO 104 NO 137 SEQ ID AGUUGCAUUCAAUGUUCUGACAACASEQ ID CGCUGCCCAAUGCCAUCCUGGAGUU NO 105 NO 138 SEQ IDUCC UGU AGA AUA CUG GCA UC SEQ ID UGUUUUUGAGGAUUGCUGAA NO 106 NO 139SEQ ID UGCAGACCUCCUGCCACCGCAGAUU SEQ ID UGUUCUGACAACAGUUUGCCGCUGCCNO 107 CA NO 140 CAAUGCCAUCCUGG SEQ ID UUGCAGACCUCCUGCCACCGCAGAU SEQ IDUUUGCCICUGCCCAAUGCCAUCCUG NO 108 UCAGGCUUC NO 557 PS305 SEQ IDUUUGCCGCUICCCAAUGCCAUCCUG SEQ ID UUUGCCGCUGCCCAIUGCCAUCCUG NO 558 NO 566SEQ ID UUUGCCGCUGCCCAAUICCAUCCUG SEQ ID UUUGCCGCUGCCCAAUGCCIUCCUG NO 559NO 567 SEQ ID UUUICCGCUGCCCAAUGCCAUCCUG SEQ ID UUUICCICUGCCCAAUGCCAUCCUGNO 560 NO 568 SEQ ID UUUGCCGCUGCCCAAUGCCAUCCUI SEQ IDUUUGCCGCUGCCCAAIGCCAUCCUG NO 561 NO 569 SEQ ID IUUGCCGCUGCCCAAUGCCAUCCUGSEQ ID UUUGCCGCUGCCCAAUGCCAICCUG NO 562 NO 570 SEQ IDUIUGCCGCUGCCCAAUGCCAUCCUG SEQ ID UUUGCCGCUGCCCAAUGCCAUCCIG NO 563 NO 571SEQ ID UUIGCCGCUGCCCAAUGCCAUCCUG SEQ ID UUUGCCGCUGCCCIAUGCCAUCCUG NO 564NO 572 SEQ ID UUUGCCGCIGCCCAAUGCCAUCCUG NO 565 DMD Gene Exon 53 SEQ IDCUCUGGCCUGUCCUAAGACCUGCUC SEQ ID CAGCUUCUUCCUUAGCUUCCAGCCA NO 141 NO 165SEQ ID UCUGGCCUGUCCUAAGACCUGCUCA SEQ ID AGCUUCUUCCUUAGCUUCCAGCCAU NO 142NO 166 SEQ ID CUGGCCUGUCCUAAGACCUGCUCAG SEQ ID GCUUCUUCCUUAGCUUCCAGCCAUUNO 143 NO 167 SEQ ID UGGCCUGUCCUAAGACCUGCUCAGC SEQ IDCUUCUUCCUUAGCUUCCAGCCAUUG NO 144 NO 168 SEQ ID GGCCUGUCCUAAGACCUGCUCAGCUSEQ ID UUCUUCCUUAGCUUCCAGCCAUUGU NO 145 NO 169 SEQ IDGCCUGUCCUAAGACCUGCUCAGCUU SEQ ID UCUUCCUUAGCUUCCAGCCAUUGUG NO 146 NO 170SEQ ID CCUGUCCUAAGACCUGCUCAGCUUC SEQ ID CUUCCUUAGCUUCCAGCCAUUGUGU NO 147NO 171 SEQ ID CUGUCCUAAGACCUGCUCAGCUUCU SEQ ID UUCCUUAGCUUCCAGCCAUUGUGUUNO 148 NO 172 SEQ ID UGUCCUAAGACCUGCUCAGCUUCUU SEQ IDUCCUUAGCUUCCAGCCAUUGUGUUG NO 149 NO 173 SEQ ID GUCCUAAGACCUGCUCAGCUUCUUCSEQ ID CCUUAGCUUCCAGCCAUUGUGUUGA NO 150 NO 174 SEQ IDUCCUAAGACCUGCUCAGCUUCUUCC SEQ ID CUUAGCUUCCAGCCAUUGUGUUGAA NO 151 NO 175SEQ ID CCUAAGACCUGCUCAGCUUCUUCCU SEQ ID UUAGCUUCCAGCCAUUGUGUUGAAU NO 152NO 176 SEQ ID CUAAGACCUGCUCAGCUUCUUCCUU SEQ ID UAGCUUCCAGCCAUUGUGUUGAAUCNO 153 NO 177 SEQ ID UAAGACCUGCUCAGCUUCUUCCUUA SEQ IDAGCUUCCAGCCAUUGUGUUGAAUCC NO 154 NO 178 SEQ ID AAGACCUGCUCAGCUUCUUCCUUAGSEQ ID GCUUCCAGCCAUUGUGUUGAAUCCU NO 155 NO 179 SEQ IDAGACCUGCUCAGCUUCUUCCUUAGC SEQ ID CUUCCAGCCAUUGUGUUGAAUCCUU NO 156 NO 180SEQ ID GACCUGCUCAGCUUCUUCCUUAGCU SEQ ID UUCCAGCCAUUGUGUUGAAUCCUUU NO 157NO 181 SEQ ID ACCUGCUCAGCUUCUUCCUUAGCUU SEQ ID UCCAGCCAUUGUGUUGAAUCCUUUANO 158 NO 182 SEQ ID CCUGCUCAGCUUCUUCCUUAGCUUC SEQ IDCCAGCCAUUGUGUUGAAUCCUUUAA NO 159 NO 183 SEQ ID CUGCUCAGCUUCUUCCUUAGCUUCCSEQ ID CAGCCAUUGUGUUGAAUCCUUUAAC NO 160 NO 184 SEQ IDUGCUCAGCUUCUUCCUUAGCUUCCA SEQ ID AGCCAUUGUGUUGAAUCCUUUAACA NO 161 NO 185SEQ ID GCUCAGCUUCUUCCUUAGCUUCCAG SEQ ID GCCAUUGUGUUGAAUCCUUUAACAU NO 162NO 186 SEQ ID CUCAGCUUCUUCCUUAGCUUCCAGC SEQ ID CCAUUGUGUUGAAUCCUUUAACAUUNO 163 NO 187 SEQ ID UCAGCUUCUUCCUUAGCUUCCAGCC SEQ IDCAUUGUGUUGAAUCCUUUAACAUUU NO 164 NO 188 DMD Gene Exon 44 SEQ IDUCAGCUUCUGUUAGCCACUG SEQ ID AGCUUCUGUUAGCCACUGAUUAAA NO 189 NO 214SEQ ID UUCAGCUUCUGUUAGCCACU SEQ ID CAGCUUCUGUUAGCCACUGAUUAA NO 190NO 215 A SEQ ID UUCAGCUUCUGUUAGCCACUG SEQ ID AGCUUCUGUUAGCCACUGAUUAAANO 191 NO 216 SEQ ID UCAGCUUCUGUUAGCCACUGA SEQ ID AGCUUCUGUUAGCCACUGAUNO 192 NO 217 SEQ ID UUCAGCUUCUGUUAGCCACUGA SEQ ID GCUUCUGUUAGCCACUGAUUNO 193 NO 218 SEQ ID UCAGCUUCUGUUAGCCACUGA SEQ ID AGCUUCUGUUAGCCACUGAUUNO 194 NO 219 SEQ ID UUCAGCUUCUGUUAGCCACUGA SEQ ID GCUUCUGUUAGCCACUGAUUANO 195 NO 220 SEQ ID UCAGCUUCUGUUAGCCACUGAU SEQ IDAGCUUCUGUUAGCCACUGAUUA NO 196 NO 221 SEQ ID UUCAGCUUCUGUUAGCCACUGAUSEQ ID GCUUCUGUUAGCCACUGAUUAA NO 197 NO 222 SEQ IDUCAGCUUCUGUUAGCCACUGAUU SEQ ID AGCUUCUGUUAGCCACUGAUUAA NO 198 NO 223SEQ ID UUCAGCUUCUGUUAGCCACUGAUU SEQ ID GCUUCUGUUAGCCACUGAUUAAA NO 199NO 224 SEQ ID UCAGCUUCUGUUAGCCACUGAUUA SEQ ID AGCUUCUGUUAGCCACUGAUUAAANO 200 NO 225 SEQ ID UUCAGCUUCUGUUAGCCACUGAUA SEQ IDGCUUCUGUUAGCCACUGAUUAAA NO 201 NO 226 SEQ ID UCAGCUUCUGUUAGCCACUGAUUAASEQ ID CCAUUUGUAUUUAGCAUGUUCCC NO 202 NO 227 SEQ IDUUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID AGAUACCAUUUGUAUUUAGC NO 203 NO 228SEQ ID UCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID GCCAUUUCUCAACAGAUCU NO 204NO 229 SEQ ID UUCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID GCCAUUUCUCAACAGAUCUGUCANO 205 NO 230 SEQ ID CAGCUUCUGUUAGCCACUG SEQ ID AUUCUCAGGAAUUUGUGUCUUUCNO 206 NO 231 SEQ ID CAGCUUCUGUUAGCCACUGAU SEQ ID UCUCAGGAAUUUGUGUCUUUCNO 207 NO 232 SEQ ID AGCUUCUGUUAGCCACUGAUU SEQ ID GUUCAGCUUCUGUUAGCCNO 208 NO 233 SEQ ID CAGCUUCUGUUAGCCACUGAUU SEQ IDCUGAUUAAAUAUCUUUAUAU C NO 209 NO 234 SEQ ID AGCUUCUGUUAGCCACUGAUUASEQ ID GCCGCCAUUUCUCAACAG NO 210 NO 235 SEQ ID CAGCUUCUGUUAGCCACUGAUUASEQ ID GUAUUUAGCAUGUUCCCA NO 211 NO 236 SEQ ID AGCUUCUGUUAGCCACUGAUUAASEQ ID CAGGAAUUUGUGUCUUUC NO 212 NO 237 SEQ ID CAGCUUCUGUUAGCCACUGAUUAASEQ ID UCAICUUCUGUUAGCCACUG NO 213 NO 575 SEQ ID UCAGCUUCUIUUAGCCACUGSEQ ID UCAGCUUCUGUUAGCCACUI NO 573 NO 576 SEQ ID UCAGCUUCUGUUAICCACUGNO 574 DMD Gene Exon 46 SEQ ID GCUUUUCUUUUAGUUGCUGCUCUUU SEQ IDCCAGGUUCAAGUGGGAUACUAGCAA NO 238 NO 265 SEQ ID CUUUUCUUUUAGUUGCUGCUCUUUUSEQ ID CAGGUUCAAGUGGGAUACUAGCAAU NO 239 NO 266 SEQ IDUUUUCUUUUAGUUGCUGCUCUUUUC SEQ ID AGGUUCAAGUGGGAUACUAGCAAUG NO 240 NO 267SEQ ID UUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID GGUUCAAGUGGGAUACUAGCAAUGU NO 241NO 268 SEQ ID UUCUUUUAGUUGCUGCUCUUUUCCA SEQ ID GUUCAAGUGGGAUACUAGCAAUGUUNO 242 NO 269 SEQ ID UCUUUUAGUUGCUGCUCUUUUCCAG SEQ IDUUCAAGUGGGAUACUAGCAAUGUUA NO 243 NO 270 SEQ ID CUUUUAGUUGCUGCUCUUUUCCAGGSEQ ID UCAAGUGGGAUACUAGCAAUGUUAU NO 244 NO 271 SEQ IDUUUUAGUUGCUGCUCUUUUCCAGGU SEQ ID CAAGUGGGAUACUAGCAAUGUUAUC NO 245 NO 272SEQ ID UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID AAGUGGGAUACUAGCAAUGUUAUCU NO 246NO 273 SEQ ID UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID AGUGGGAUACUAGCAAUGUUAUCUGNO 247 NO 274 SEQ ID UAGUUGCUGCUCUUUUCCAGGUUCA SEQ IDGUGGGAUACUAGCAAUGUUAUCUGC NO 248 NO 275 SEQ ID AGUUGCUGCUCUUUUCCAGGUUCAASEQ ID UGGGAUACUAGCAAUGUUAUCUGCU NO 249 NO 276 SEQ IDGUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID GGGAUACUAGCAAUGUUAUCUGCUU NO 250 NO 277SEQ ID UUGCUGCUCUUUUCCAGGUUCAAGU SEQ ID GGAUACUAGCAAUGUUAUCUGCUUC NO 251NO 278 SEQ ID UCCUGCUCUUUUCCAGGUUCAAGUG SEQ ID GAUACUAGCAAUGUUAUCUGCUUCCNO 252 NO 279 SEQ ID GCUGCUCUUUUCCAGGUUCAAGUGG SEQ IDAUACUAGCAAUGUUAUCUGCUUCCU NO 253 NO 280 SEQ ID CUGCUCUUUUCCAGGUUCAAGUGGGSEQ ID UACUAGCAAUGUUAUCUGCUUCCUC NO 254 NO 281 SEQ IDUGCUCUUUUCCAGGUUCAAGUGGGA SEQ ID ACUAGCAAUGUUAUCUGCUUCCUCC NO 255 NO 282SEQ ID GCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID CUAGCAAUGUUAUCUGCUUCCUCCA NO 256NO 283 SEQ ID CUCUUUUCCAGGUUCAAGUGGGAUA SEQ ID UAGCAAUGUUAUCUGCUUCCUCCAANO 257 NO 284 SEQ ID UCUUUUCCAGGUUCAAGUGGGAUAC SEQ IDAGCAAUGUUAUCUGCUUCCUCCAAC NO 258 NO 285 SEQ ID UCUUUUCCAGGUUCAAGUGGSEQ ID GCAAUGUUAUCUGCUUCCUCCAACC NO 259 NO 286 SEQ IDCUUUUCCAGGUUCAAGUGGGAUACU SEQ ID CAAUGUUAUCUGCUUCCUCCAACCA NO 260 NO 287SEQ ID UUUUCCAGGUUCAAGUGGGAUACUA SEQ ID AAUGUUAUCUGCUUCCUCCAACCAU NO 261NO 288 SEQ ID UUUCCAGGUUCAAGUGGGAUACUAG SEQ ID AUGUUAUCUGCUUCCUCCAACCAUANO 262 NO 289 SEQ ID UUCCAGGUUCAAGUGGGAUACUAGC SEQ IDUGUUAUCUGCUUCCUCCAACCAUAA NO 263 NO 290 SEQ ID UCCAGGUUCAAGUGGGAUACUAGCANO 264 DMD Gene Exon 52 SEQ ID AGCCUCUUGAUUGCUGGUCUUGUUU SEQ IDUUGGGCAGCGGUAAUGAGUUCUUCC NO 291 NO 326 SEQ ID GCCUCUUGAUUGCUGGUCUUGUUUUSEQ ID UGGGCAGCGGUAAUGAGUUCUUCCA NO 292 NO 327 SEQ IDCCUCUUGAUUGCUGGUCUUGUUUUU SEQ ID GGGCAGCGGUAAUGAGUUCUUCCAA NO 293 NO 328SEQ ID CCUCUUGAUUGCUGGUCUUG SEQ ID GGCAGCGGUAAUGAGUUCUUCCAAC NO 294NO 329 SEQ ID CUCUUGAUUGCUGGUCUUGUUUUUC SEQ ID GCAGCGGUAAUGAGUUCUUCCAACUNO 295 NO 330 SEQ ID UCUUGAUUGCUGGUCUUGUUUUUCA SEQ IDCAGCGGUAAUGAGUUCUUCCAACUG NO 296 NO 331 SEQ ID CUUGAUUGCUGGUCUUGUUUUUCAASEQ ID AGCGGUAAUGAGUUCUUCCAACUGG NO 297 NO 332 SEQ IDUUGAUUGCUGGUCUUGUUUUUCAAA SEQ ID GCGGUAAUGAGUUCUUCCAACUGGG NO 298 NO 333SEQ ID UGAUUGCUGGUCUUGUUUUUCAAAU SEQ ID CGGUAAUGAGUUCUUCCAACUGGGG NO 299NO 334 SEQ ID GAUUGCUGGUCUUGUUUUUCAAAUU SEQ ID GGUAAUGAGUUCUUCCAACUGGGGANO 300 NO 335 SEQ ID GAUUGCUGGUCUUGUUUUUC SEQ ID GGUAAUGAGUUCUUCCAACUGGNO 301 NO 336 SEQ ID AUUGCUGGUCUUGUUUUUCAAAUUU SEQ IDGUAAUGAGUUCUUCCAACUGGGGAC NO 302 NO 337 SEQ ID UUGCUGGUCUUGUUUUUCAAAUUUUSEQ ID UAAUGAGUUCUUCCAACUGGGGACG NO 303 NO 338 SEQ IDUGCUGGUCUUGUUUUUCAAAUUUUG SEQ ID AAUGAGUUCUUCCAACUGGGGACGC NO 304 NO 339SEQ ID GCUGGUCUUGUUUUUCAAAUUUUGG SEQ ID AUGAGUUCUUCCAACUGGGGACGCC NO 305NO 340 SEQ ID CUGGUCUUGUUUUUCAAAUUUUGGG SEQ ID UGAGUUCUUCCAACUGGGGACGCCUNO 306 NO 341 SEQ ID UGGUCUUGUUUUUCAAAUUUUGGGC SEQ IDGAGUUCUUCCAACUGGGGACGCCUC NO 307 NO 342 SEQ ID GGUCUUGUUUUUCAAAUUUUGGGCASEQ ID AGUUCUUCCAACUGGGGACGCCUCU NO 308 NO 343 SEQ IDGUCUUGUUUUUCAAAUUUUGGGCAG SEQ ID GUUCUUCCAACUGGGGACGCCUCUG NO 309 NO 344SEQ ID UCUUGUUUUUCAAAUUUUGGGCAGC SEQ ID UUCUUCCAACUGGGGACGCCUCUGU NO 310NO 345 SEQ ID CUUGUUUUUCAAAUUUUGGGCAGCG SEQ ID UCUUCCAACUGGGGACGCCUCUGUUNO 311 NO 346 SEQ ID UUGUUUUUCAAAUUUUGGGCAGCGG SEQ IDCUUCCAACUGGGGACGCCUCUGUUC NO 312 NO 347 SEQ ID UGUUUUUCAAAUUUUGGGCAGCGGUSEQ ID UUCCAACUGGGGACGCCUCUGUUCC NO 313 NO 348 SEQ IDGUUUUUCAAAUUUUGGGCAGCGGUA SEQ ID UCCAACUGGGGACGCCUCUGUUCCA NO 314 NO 349SEQ ID UUUUUCAAAUUUUGGGCAGCGGUAA SEQ ID CCAACUGGGGACGCCUCUGUUCCAA NO 315NO 350 SEQ ID UUUUCAAAUUUUGGGCAGCGGUAAU SEQ ID CAACUGGGGACGCCUCUGUUCCAAANO 316 NO 351 SEQ ID UUUCAAAUUUUGGGCAGCGGUAAUG SEQ IDAACUGGGGACGCCUCUGUUCCAAAU NO 317 NO 352 SEQ ID UUCAAAUUUUGGGCAGCGGUAAUGASEQ ID ACUGGGGACGCCUCUGUUCCAAAUC NO 318 NO 353 SEQ IDUCAAAUUUUGGGCAGCGGUAAUGAG SEQ ID CUGGGGACGCCUCUGUUCCAAAUCC NO 319 NO 354SEQ ID CAAAUUUUGGGCAGCGGUAAUGAGU SEQ ID UGGGGACGCCUCUGUUCCAAAUCCU NO 320NO 355 SEQ ID AAAUUUUGGGCAGCGGUAAUGAGUU SEQ ID GGGGACGCCUCUGUUCCAAAUCCUGNO 321 NO 356 SEQ ID AAUUUUGGGCAGCGGUAAUGAGUUC SEQ IDGGGACGCCUCUGUUCCAAAUCCUGC NO 322 NO 357 SEQ ID AUUUUGGGCAGCGGUAAUGAGUUCUSEQ ID GGACGCCUCUGUUCCAAAUCCUGCA NO 323 NO 358 SEQ IDUUUUGGGCAGCGGUAAUGAGUUCUU SEQ ID GACGCCUCUGUUCCAAAUCCUGCAU NO 324 NO 359SEQ ID UUUGGGCAGCGGUAAUGAGUUCUUC NO 325 DMD Gene Exon 50 SEQ IDCCAAUAGUGGUCAGUCCAGGAGCUA SEQ ID CUAGGUCAGGCUGCUUUGCCCUCAG NO 360 NO 386SEQ ID CAAUAGUGGUCAGUCCAGGAGCUAG SEQ ID UAGGUCAGGCUGCUUUGCCCUCAGC NO 361NO 387 SEQ ID AAUAGUGGUCAGUCCAGGAGCUAGG SEQ ID AGGUCAGGCUGCUUUGCCCUCAGCUNO 362 NO 388 SEQ ID AUAGUGGUCAGUCCAGGAGCUAGGU SEQ IDGGUCAGGCUGCUUUGCCCUCAGCUC NO 363 NO 389 SEQ ID AUAGUGGUCAGUCCAGGAGCUSEQ ID GUCAGGCUGCUUUGCCCUCAGCUCU NO 364 NO 390 SEQ IDUAGUGGUCAGUCCAGGAGCUAGGUC SEQ ID UCAGGCUGCUUUGCCCUCAGCUCUU NO 365 NO 391SEQ ID AGUGGUCAGUCCAGGAGCUAGGUCA SEQ ID CAGGCUGCUUUGCCCUCAGCUCUUG NO 366NO 392 SEQ ID GUGGUCAGUCCAGGAGCUAGGUCAG SEQ ID AGGCUGCUUUGCCCUCAGCUCUUGANO 367 NO 393 SEQ ID UGGUCAGUCCAGGAGCUAGGUCAGG SEQ IDGGCUGCUUUGCCCUCAGCUCUUGAA NO 368 NO 394 SEQ ID GGUCAGUCCAGGAGCUAGGUCAGGCSEQ ID GCUGCUUUGCCCUCAGCUCUUGAAG NO 369 NO 395 SEQ IDGUCAGUCCAGGAGCUAGGUCAGGCU SEQ ID CUGCUUUGCCCUCAGCUCUUGAAGU NO 370 NO 396SEQ ID UCAGUCCAGGAGCUAGGUCAGGCUG SEQ ID UGCUUUGCCCUCAGCUCUUGAAGUA NO 371NO 397 SEQ ID CAGUCCAGGAGCUAGGUCAGGCUGC SEQ ID GCUUUGCCCUCAGCUCUUGAAGUAANO 372 NO 398 SEQ ID AGUCCAGGAGCUAGGUCAGGCUGCU SEQ IDCUUUGCCCUCAGCUCUUGAAGUAAA NO 373 NO 399 SEQ ID GUCCAGGAGCUAGGUCAGGCUGCUUSEQ ID UUUGCCCUCAGCUCUUGAAGUAAAC NO 374 NO 400 SEQ IDUCCAGGAGCUAGGUCAGGCUGCUUU SEQ ID UUGCCCUCAGCUCUUGAAGUAAACG NO 375 NO 401SEQ ID CCAGGAGCUAGGUCAGGCUGCUUUG SEQ ID UGCCCUCAGCUCUUGAAGUAAACGG NO 376NO 402 SEQ ID CAGGAGCUAGGUCAGGCUGCUUUGC SEQ ID GCCCUCAGCUCUUGAAGUAAACGGUNO 377 NO 403 SEQ ID AGGAGCUAGGUCAGGCUGCUUUGCC SEQ IDCCCUCAGCUCUUGAAGUAAACGGUU NO 378 NO 404 SEQ ID GGAGCUAGGUCAGGCUGCUUUGCCCSEQ ID CCUCAGCUCUUGAAGUAAAC NO 379 NO 405 SEQ IDGAGCUAGGUCAGGCUGCUUUGCCCU SEQ ID CCUCAGCUCUUGAAGUAAACG NO 380 NO 406SEQ ID AGCUAGGUCAGGCUGCUUUGCCCUC SEQ ID CUCAGCUCUUGAAGUAAACG NO 381NO 407 SEQ ID GCUAGGUCAGGCUGCUUUGCCCUCA SEQ ID CCUCAGCUCUUGAAGUAAACGGUUUNO 382 NO 408 SEQ ID CUCAGCUCUUGAAGUAAACGGUUUA SEQ IDUCAGCUCUUGAAGUAAACGGUUUAC NO 383 NO 409 SEQ ID CAGCUCUUGAAGUAAACGGUUUACCSEQ ID AGCUCUUGAAGUAAACGGUUUACCG NO 384 NO 410 SEQ IDGCUCUUGAAGUAAACGGUUUACCGC SEQ ID CUCUUGAAGUAAACGGUUUACCGCC NO 385 NO 411DMD Gene Exon 43 SEQ ID CCACAGGCGUUGCACUUUGCAAUGC SEQ ID UCUUCUUGCUAUGAAUAAUGUCAAU NO 412 NO 443 SEQ ID CACAGGCGUUGCACUUUGCAAUGCUSEQ ID CUUCUUGCUAUGAAUAAUGUCAAUC NO 413 NO 444 SEQ IDACAGGCGUUGCACUUUGCAAUGCUG SEQ ID UUCUUGCUAUGAAUAAUGUCAAUCC NO 414 NO 445SEQ ID CAGGCGUUGCACUUUGCAAUGCUGC SEQ ID UCUUGCUAUGAAUAAUGUCAAUCCG NO 415NO 446 SEQ ID AGGCGUUGCACUUUGCAAUGCUGCU SEQ ID CUUGCUAUGAAUAAUGUCAAUCCGANO 416 NO 447 SEQ ID GGCGUUGCACUUUGCAAUGCUGCUG SEQ IDUUGCUAUGAAUAAUGUCAAUCCGAC NO 417 NO 448 SEQ ID GCGUUGCACUUUGCAAUGCUGCUGUSEQ ID UGCUAUGAAUAAUGUCAAUCCGACC NO 418 NO 449 SEQ IDCGUUGCACUUUGCAAUGCUGCUGUC SEQ ID GCUAUGAAUAAUGUCAAUCCGACCU NO 419 NO 450SEQ ID CGUUGCACUUUGCAAUGCUGCUG SEQ ID CUAUGAAUAAUGUCAAUCCGACCUG NO 420NO 451 SEQ ID GUUGCACUUUGCAAUGCUGCUGUCU SEQ ID UAUGAAUAAUGUCAAUCCGACCUGANO 421 NO 452 SEQ ID UUGCACUUUGCAAUGCUGCUGUCUU SEQ IDAUGAAUAAUGUCAAUCCGACCUGAG NO 422 NO 453 SEQ ID UGCACUUUGCAAUGCUGCUGUCUUCSEQ ID UGAAUAAUGUCAAUCCGACCUGAGC NO 423 NO 454 SEQ IDGCACUUUGCAAUGCUGCUGUCUUCU SEQ ID GAAUAAUGUCAAUCCGACCUGAGCU NO 424 NO 455SEQ ID CACUUUGCAAUGCUGCUGUCUUCUU SEQ ID AAUAAUGUCAAUCCGACCUGAGCUU NO 425NO 456 SEQ ID ACUUUGCAAUGCUGCUGUCUUCUUG SEQ ID AUAAUGUCAAUCCGACCUGAGCUUUNO 426 NO 457 SEQ ID CUUUGCAAUGCUGCUGUCUUCUUGC SEQ IDUAAUGUCAAUCCGACCUGAGCUUUG NO 427 NO 458 SEQ ID UUUGCAAUGCUGCUGUCUUCUUGCUSEQ ID AAUGUCAAUCCGACCUGAGCUUUGU NO 428 NO 459 SEQ IDUUGCAAUGCUGCUGUCUUCUUGCUA SEQ ID AUGUCAAUCCGACCUGAGCUUUGUU NO 429 NO 460SEQ ID UGCAAUGCUGCUGUCUUCUUGCUAU SEQ ID UGUCAAUCCGACCUGAGCUUUGUUG NO 430NO 461 SEQ ID GCAAUGCUGCUGUCUUCUUGCUAUG SEQ ID GUCAAUCCGACCUGAGCUUUGUUGUNO 431 NO 462 SEQ ID CAAUGCUGCUGUCUUCUUGCUAUGA SEQ IDUCAAUCCGACCUGAGCUUUGUUGUA NO 432 NO 463 SEQ ID AAUGCUGCUGUCUUCUUGCUAUGAASEQ ID CAAUCCGACCUGAGCUUUGUUGUAG NO 433 NO 464 SEQ IDAUGCUGCUGUCUUCUUGCUAUGAAU SEQ ID AAUCCGACCUGAGCUUUGUUGUAGA NO 434 NO 465SEQ ID UGCUGCUGUCUUCUUGCUAUGAAUA SEQ ID AUCCGACCUGAGCUUUGUUGUAGAC NO 435NO 466 SEQ ID GCUGCUGUCUUCUUGCUAUGAAUAA SEQ ID UCCGACCUGAGCUUUGUUGUAGACUNO 436 NO 467 SEQ ID CUGCUGUCUUCUUGCUAUGAAUAAU SEQ IDCCGACCUGAGCUUUGUUGUAGACUA NO 437 NO 468 SEQ ID UGCUGUCUUCUUGCUAUGAAUAAUGSEQ ID CGACCUGAGCUUUGUUGUAG NO 438 NO 469 SEQ IDGCUGUCUUCUUGCUAUGAAUAAUGU SEQ ID CGACCUGAGCUUUGUUGUAGACUAU NO 439 NO 470SEQ ID CUGUCUUCUUGCUAUGAAUAAUGUC SEQ ID GACCUGAGCUUUGUUGUAGACUAUC NO 440NO 471 SEQ ID UGUCUUCUUGCUAUGAAUAAUGUCA SEQ ID ACCUGAGCUUUGUUGUAGACUAUCANO 441 NO 472 SEQ ID GUCUUCUUGCUAUGAAUAAUGUCAA SEQ IDCCUGAGCUUUGUUGUAGACUAUC NO 442 NO 473 DMD Gene Exon 6 SEQ IDCAUUUUUGACCUACAUGUGG SEQ ID AUUUUUGACCUACAUGGGAAA G NO 474 NO 479 SEQ IDUUUGACCUACAUGUGGAAAG SEQ ID UACGAGUUGAUUGUCGGACCCAG NO 475 NO 480 SEQ IDUACAUUUUUGACCUACAUGUGGAAA SEQ ID GUGGUCUCCUUACCUAUGACUGUGG NO 476 GNO 481 SEQ ID GGUCUCCUUACCUAUGA SEQ ID UGUCUCAGUAAUCUUCUUACCUAU NO 477NO 482 SEQ ID UCUUACCUAUGACUAUGGAUGAGA NO 478 DMD Gene Exon 7 SEQ IDUGCAUGUUCCAGUCGUUGUGUGG SEQ ID AUUUACCAACCUUCAGGAUCGAGUA NO 483 NO 485SEQ ID CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID GGCCUAAAACACAUACACAUA NO 484NO 486 DMD Gene Exon 8 SEQ ID GAUAGGUGGUAUCAACAUCUGUAA SEQ IDUGUUGUUGUUUAUGCUCAUU NO 487 NO 490 SEQ ID GAUAGGUGGUAUCAACAUCUG SEQ IDGUACAUUAAGAUGGACUUC NO 488 NO 491 SEQ ID CUUCCUGGAUGGCUUGAAU NO 489DMD Gene Exon 55 SEQ ID CUGUUGCAGUAAUCUAUGAG SEQ IDUGCCAUUGUUUCAUCAGCUCUUU NO 492 NO 495 SEQ ID UGCAGUAAUCUAUGAGUUUC SEQ IDUCCUGUAGGACAUUGGCAGU NO 493 NO 496 SEQ ID GAGUCUUCUAGGAGCCUU SEQ IDCUUGGAGUCUUCUAGGAGCC NO 494 NO 497 DMD Gene Exon 2 SEQ IDCCAUUUUGUGAAUGUUUUCUUUUG SEQ ID GAAAAUUGUGCAUUUACCCAUUUU NO 498 AACAUCNO 500 SEQ ID CCCAUUUUGUGAAUGUUUUCUUUU SEQ ID UUGUGCAUUUACCCAUUUUGUGNO 499 NO 501 DMD Gene Exon 11 SEQ ID CCCUGAGGCAUUCCCAUCUUGAAU SEQ ID CUUGAAUUUAGGAGAUUCAUCUG NO 502 NO 504 SEQ ID AGGACUUACUUGCUUUGUUU SEQ IDCAUCUUCUGAUAAUUUUCCUGUU NO 503 NO 505 DMD Gene Exon 17 SEQ IDCCAUUACAGUUGUCUGUGUU SEQ ID UAAUCUGCCUCUUCUUUUGG NO 506 NO 508 SEQ IDUGACAGCCUGUGAAAUCUGUGAG NO 507 DMD Gene Exon 19 SEQ IDCAGCAGUAGUUGUCAUCUGC SEQ ID GCCUGAGCUGAUCUGCUGGCAUCUUG NO 509 NO 511CAGUU SEQ ID GCCUGAGCUGAUCUGCUGGCAUCUU SEQ ID UCUGCUGGCAUCUUGC NO 510 GCNO 512 DMD Gene Exon 21 SEQ ID GCCGGUUGACUUCAUCCUGUGC SEQ IDCUGCAUCCAGGAACAUGGGUCC NO 513 NO 516 SEQ ID GUCUGCAUCCAGGAACAUGGGUCSEQ ID GUUGAAGAUCUGAUAGCCGGUUGA NO 514 NO 517 SEQ IDUACUUACUGUCUGUAGCUCUUUCU NO 515 DMD Gene Exon 57 SEQ IDUAGGUGCCUGCCGGCUU SEQ ID CUGAACUGCUGGAAAGUCGCC NO 518 NO 520 SEQ IDUUCAGCUGUAGCCACACC SEQ ID CUGGCUUCCAAAUGGGACCUGAAAAA NO 519 NO 521 GAACDMD Gene Exon 59 SEQ ID CAAUUUUUCCCACUCAGUAUU NO 522 SEQ IDUCCUCAGGAGGCAGCUCUAAAU SEQ ID UUGAAGUUCCUGGAGUCUU NO 524 NO 523DMD Gene Exon 62 SEQ ID UGGCUCUCUCCCAGGG SEQ ID GGGCACUUUGUUUGGCG NO 525NO 527 SEQ ID GAGAUGGCUCUCUCCCAGGGACCCU NO 526 GG DMD Gene Exon 63SEQ ID GGUCCCAGCAAGUUGUUUG SEQ ID GUAGAGCUCUGUCAUUUUGGG NO 528 NO 530SEQ ID UGGGAUGGUCCCAGCAAGUUGUUUG NO 529 DMD Gene Exon 65 SEQ IDGCUCAAGAGAUCCACUGCAAAAAAC SEQ ID UCUGCAGGAUAUCCAUGGGCUGGUC NO 531 NO 533SEQ ID GCCAUACGUACGUAUCAUAAACAUU NO 532 C DMD Gene Exon 66 SEQ IDGAUCCUCCCUGUUCGUCCCCUAUUA NO 534 UG DMD Gene Exon 69 SEQ IDUGCUUUAGACUCCUGUACCUGAUA NO 535 DMD Gene Exon 75 SEQ IDGGCGGCCUUUGUGUUGAC SEQ ID CCUUUAUGUUCGUGCUGCU NO 536 NO 538 SEQ IDGGACAGGCCUUUAUGUUCGUGCUGC NO 537Human IGF-1 Isoform 4 amino acid sequence SEQ ID NO 577:MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM

What is claimed is:
 1. An oligonucleotide consisting of the basesequence of any one of SEQ ID NO: 141, 142 or 144-188, wherein at leastone guanosine base in the sequence is substituted with an inosine base,and wherein said oligonucleotide comprises a modification and inducesskipping of exon 53 of human dystrophin pre-mRNA.
 2. The oligonucleotideof claim 1, wherein one to four guanosine bases in the sequence aresubstituted with an inosine base.
 3. The oligonucleotide of claim 1,wherein the modification is a base and/or sugar modification.
 4. Theoligonucleotide of claim 3, wherein the oligonucleotide is a 2′-O-methylphosphorothioate oligonucleotide.
 5. The oligonucleotide of claim 3,wherein the oligonucleotide is a peptide nucleic acid.
 6. Theoligonucleotide of claim 3, wherein the oligonucleotide is aphosphorodiamidate morpholino oligomer.
 7. A pharmaceutical composition,comprising the oligonucleotide of claim 1 and a pharmaceuticallyacceptable carrier.
 8. A method for inducing skipping of exon 53 ofhuman dystrophin pre-mRNA in a human subject, comprising administeringthe oligonucleotide of claim 1 to the human subject in an amount and fora time which is effective to induce exon skipping.
 9. A method foralleviating one or more symptom(s) of Duchenne Muscular Dystrophy orBecker Muscular Dystrophy in a human subject, comprising administeringto the human subject the oligonucleotide of claim 1.